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1916 


A  LABORATORY  MANUAL 
OF  BIOLOGICAL  CHEMISTRY 


m 


LABORATORY  MANUAL 
OF  BIOLOGICAL  CHEMISTRY 


. 


LABORATORY  MANUAL  OF 
BIOLOGICAL    CHEMISTRY 


WITH   SUPPLEMENT 


BY 
OTTO   FOLIN 

Hamilton  Kuhn  Professor  of  Biological  Chemistry  in  Harvard  Medical  School 


NEW  YORK  AND    LONDON 
D.  APPLETON  AND  COMPANY 

1919 


p 

H. 

d-C 

BIOLOGY 

LIBRARY 

6 


COPYRIGHT,    1916,    1919,    BY 

D.  APPLETON  AND  COMPANY 


PRINTEP  IN  THE    UNITED  STATES  O?  AMERICA 


PREFACE  TO  SECOND  EDITION 

For  this  edition  the  manual  has  been  very  largely  rewritten. 
Much  painstaking  research  is  represented  in  the  revised  analytical 
methods  here  given  for  the  first  time  in  book  fqrm,  and  if  the 
directions  are  followed  these  methods  give  reliable  results. 

Much  valuable  assistance  has  been  received  from  Dr.  C.  H. 
Fiske  in  connection  with  this  revision. 

OTTO  FOLIN 

BOSTON 


PREFACE 

This  manual  of  biological  chemistry  for  medical  students  in 
Harvard  Medical  School  has  been  revised  annually  for  the  past 
seven  years,  and  it  is  believed  now  to  meet  our  needs  sufficiently 
well  to  warrant  publication. 

For  many  years  I  have  been  interested  in  the  development  of 
analytical  methods  applicable  to  metabolism  investigations.  The 
most  serviceable  of  my  older  methods  and  some  of  the  newer 
methods  have  been  taught  to  our  medical  students ;  these  are  de- 
scribed in  the  main  body  of  the  manual.  Others  not  heretofore 
included  have  been  incorporated  in  the  supplement,  so  that  nearly 
all  the  newer  methods  devised  in  the  department  are  now  de- 
scribed in  this  manual. 

In  connection  with  the  revisions  referred  to  above  I  am  in- 
debted for  valuable  help  to  W.  R.  Bloor,  W.  Denis,  C.  J.  Farmer, 
L.  J.  Morris,  F.  B.  Kingsbury,  F.  S.  Hammett,  R.  D.  Bell,  and 
C.  H.  Fiske,  as  well  as  to  my  older  friend,  P.  A.  Shaffer. 

OTTO  FOLIN 
BOSTON 


TABLE  OF  CONTENTS 

CHAPTER  PAGE 

I    ACIDIMETRY,    ALKALIMETRY,    NITROGEN    DETERMINATIONS  i 

II     CATALYSIS,  CATALYZERS,  FERMENTS 33 

III  FATS 39 

IV  CARBOHYDRATES .47 

V    PROTEINS *       ...  71 

VI    URINE  ANALYSIS  AND  METABOLISM 89 

VII     BLOOD .       .  135 

VIII     MILK 139 

IX     BONE 143 

X     BILE 145 

SUPPLEMENT 

URINE 151 

BLOOD 179 


LABORATORY  MANUAL  OP  BIO 
LOGICAL  CHEMISTRY 

PART  I 
ACIDIMETRY,  ALKALIMETRY,  NITROGEN  DETERMINATION 

Equivalent  and  Normal  Solutions. — Since  the  molecular  weight 
of  sodium  hydroxid  (NaOH)  is  40  and  that  of  hydrochloric  acid 
(HC1)  is  36.46,  it  follows  that  40  g.  of  the  former  contain  the 
same  number  of  molecules  as  36.46  g.  of  the  latter.  If  40  g.  of 
sodium  hydroxid  and  36.46  g.  of  hydrochloric  acid  are  each  dis- 
solved in  pure  water  sufficient  to  make  one  liter  of  solution,  each 
liter  will  contain  the  same  number  of  dissolved  molecules. 

It  will  take  a  little  less  than  one  liter  of  water  to  make  a  liter  of 
solution  because  the  dissolved  substance  takes  up  some  space.  A  nor- 
mal sodium  hydroxid  solution  contains  four  per  cent,  of  sodium 
hydroxid.  By  per  cent,  in  the  case  of  solutions  is  usually  meant  the 
amount  of  substance  present  in  100  c.c.  of  solution. 

Mixing  equal  volumes  of  two  such  solutions  is,  therefore,  the 
same  as  bringing  together  practically  the  same  number  of  the 
two  kinds  of  molecules,  and  the  result  is  -the  instantaneous  and 
essentially  complete  transformation  into  sodium  chlorid  (and 
water). 

X  NaOH  +  X  HC1  =  X  NaCl  +  X  H2O 

If  either  or  both  of  the  solutions  should  first  be  diluted  with  a 
considerable  bulk  of,  pure  water,  the  result  on  mixing  the  two 
would  be  the  same,  for  the  extra  amount  of  water  present  takes 
no  part  in  the  reaction  (except  to  the  extent  of  absorbing  a  part 
of  the  heat  set  free). 

The  two  solutions  are  equivalent.  They  also  happen  to  be  nor- 
mal solutions.  Tire  hydrochloric  acid  is  normal  because  it  con- 

1 


tains  i  g.  of  active  or  replaceable  hydrogen  per  liter'  of  solution, 
and  not  because  it  contains  the  same  number  of  grams  of  HC1 
per  liter  as  there  are  units  in  the  molecular  weight.  The  sodium 
hydroxid  solution  is  normal  because  it  is  equivalent  to  a  solution 
containing  one  gram  of  replaceable  hydrogen  per  liter. 

The  molecular  weight  of  sulphuric  acid  is  98.  A  sulphuric  acid 
solution  containing  exactly  98  g.  per  liter  contains,  therefore,  the 
same  number  of  molecules  per  unit  volume  as  the  sodium  hy- 
droxid solution  containing  40  g.  per  liter.  But  one  molecule  of 
sulphuric  acid  requires  two  molecules  of  sodium  hydroxid  for  the 
formation  of  the  neutral  salt,  sodium  sulphate,  because  the  sul- 
phuric acid  molecule  has  two  replaceable  hydrogen  atoms.  The 
solutions  are  not  equivalent,  for  the  sulphuric  acid  contains  2  g. 
active  hydrogen  per  liter.  It  is  exactly  twice  as  strong  as  the 
sodic  hydrate  solution;  it  is  a  2  normal -solution. 

On  the  basis  of  the  above  description  of  what  constitutes  a  normal 
solution,  Calculate  the  number  of  grams  per  liter  in  tenth  normal  sul- 
phuric acid  (.iN  H2SO4),  fifth  normal  hydrochloric  acid  (.2N  HC1), 
half  normal  oxalic  acid  (-5N  C2H2O4,  2H2O),  fourth  normal  acetic 
acid  OsN  CH3COOH),  half  normal  sodic  hydrate  (.5N  NaOH), 
twentieth  normal  barium  hydrate  (.05N  Ba(OH)2),  fifth  normal 
ammonium  hydrate  (.2N  NH4OH). 

Atomic  weights  of  some  of  the  more  important  elements :  Arsenic 
(As)  74.96,  Barium  (Ba)  137.37,  Bromin  (Br)  79.92,  Calcium  (Ca) 
40.09,  Carbon  (C)  12,  Chlorin  (Cl)  35.46,  Copper  (Cu)  63.57, 
Hydrogen  (H)  1.008,  lodin  (I)  126.92,  Iron  (Fe)  55.85,  Lead  (Pb) 
207.1,  Magnesium  (Mg)  24.32,  Manganese  (Mn)  54.93,  Mercury 
(Hg)  200.6,  Nitrogen  (N)  14.01,  Oxygen  (O)  16,  Phosphorus  (P) 
31.04,  Potassium  (K)  39.1,  Sulphur  (S)  32.07,  Tin  (Sn)  119, 
Tungsten  (Wo)  184,  Uranium  (U)  238.5,  Zinc  (Zn)  65.37. 

The  same  description  of  normal  solutions  applies  to  other  sub- 
stances than  acids  and  alkalis,  as,  for  example,  reducing  and  oxi- 
dizing substances  such  as  potassium  permanganate,  potassium  bi- 
chromate, iodin,  cupric  hydrate,  stannous  chlorid.  A  normal  so- 
lution is  here  one  capable  of  liberating  I  g.  of  reducing  hydrogen 
(or  of  giving  off  exactly  sufficient  oxygen  to  oxidize  one  gram  of 
hydrogen)  per  liter.  Potassium  permanganate,  for  example,  in 
the  presence  of  sulphuric  acid  and  some  easily  oxidizable  sub- 
stance is  decomposed  as  follows : 

2KMnO4  +  3H2SO4  =  K2SO4  +  2MnSO4  +  3H2O  +  50. 

3 


As  the  two  permanganate  molecules  liberate  oxygen  enough  for 
ten  hydrogen  atoms  it  takes  only  one  fiftieth  of  the  molecular 
weight  expressed  in  grams  (3.161  g.)  to  make  one  liter  of  tenth 
normal  solution. 

The  calculation  of  what  constitutes  normal  or  equivalent  solu- 
tions of  any  reagent  is  not  very  difficult  provided  the  equation 
representing  the  chemical  reaction  involved  is  thoroughly  clear. 

To  determine  whether  a  given  unknown  solution  is  acid  or 
alkaline  it  is  usually  sufficient  to  dip  a  piece  of  delicate  violet 
colored  litmus  paper  into  it.  (If  the  solution  is  acid  the  test 
paper  turns  red ;  if  alkaline  it  turns  blue.)  Litmus,  the  substance 
with  which  the  paper  has  been  impregnated,  is  a  complex  organic 
product,  and  is  one  of  the  most  familiar  representatives  of  a 
most  useful  class  of  organic  compounds  which  are  so  sensitive 
to  acids  or  alkalis,  or  both,  that  they  clearly  and  unmistakably  in- 
dicate the  presence  of  free  acid  or  alkali  even  when  the  amounts 
present  are  so  small  as  to  be  practically  unweighable.  By  means 
of  such  indicators  and  accurate  measuring  instruments  (measur- 
ing flasks,  burets,  and  pipets),  it  becomes  a  simple  matter  to 
determine  (by  titration)  the  relative  concentration  or  equivalence 
of  acid  and  alkaline  solutions.  By  their  help  it  is  possible  to  pre- 
pare with  very  little  labor  normal  or  tenth  normal  solutions,  even 
of  acids  or  alkalis  which  cannot  be  weighed  on  the  balance,  as  for 
example,  hydrochloric  acid  and  ammonia,  both  of  which  are  gases. 
Before  this  can  be  done  we  must,  however,  possess  one  normal 
or  standard  solution  prepared  from  some  substance  which  can 
be  weighed. 

Volumetric  analysis  consists  of  measuring  the  value  of  an  un- 
known solution  in  terms  of  another  the  value  of  which  is  known 
(titration).  The  known  solution  is  prepared  directly  or  indirect- 
ly by  the  help  of  the  analytical  balance,  and  the  first  step  in  any 
kind  of  volumetric  analysis  is  the  preparation  of  the  standard 
solution  by  means  of  which  the  values  of  others  are  to  be  deter- 
mined. 


Every  student  who  has  had  no  experience  in  the  use  of  the  ana- 
lytical balance  must  consult  the  instructor  before  proceeding.  He 
should  also  ask  for  instruction  as  to  the  proper  use  of  measuring 
flasks,  pipets,  and  burets  before  using  them.  He  must  particularly 
learn  when  the  presence  of  unmeasured  quantities  of  water  does  not 
interfere  with  the  accuracy  of  the  work  and  when  a  single  drop  of 

5 


unmeasured  water  introduces  a  perceptible  error.  (See  Button's 
Volumetric  Analysis,  Part  i — "Instruments  and  Apparatus.") 

All  the  common  mineral  acids  and  strong  alkalis  contain  so 
much  water  that  it  is  in  practice  not  feasible  to  weigh  out  with 
sufficient  accuracy  the  theoretical  quantity  required  for  a  standard 
solution  of  acid  or  alkali.  The  carbonates  of  sodium  or  calcium 
(or  the  carbonates  of  sodium  or  potassium,  obtained  by  ignition 
of  the  corresponding  oxalates)  give  exceedingly  accurate  results. 
Oxalic  acid  is  very  serviceable  as  starting  material  for  the  prepa- 
ration of  standardized  solutions  of  acids  and  alkalis  if  it  is  pure 
and  has  lost  none  of  its  water  of  crystallization. 

1.  Calibrations. — The  volumetric  ware  now  available  is  not 
always  accurate  enough  for  the  work  of  this  course.  Some 
approximate  calibrations  are  therefore  necessary. 

At  least  i  pipet  should  be  calibrated  by  weight.  Clean  a  20 
c.c.  pipet  with  '^cleaning  fluid"  and  rinse.  Weigh  a  clean  and 
empty  but  not  necessarily  dry  100  c.c.  volumetric  flask  within 
an  accuracy  of  2  mg.  Fill  the  pipet,  adjust  the  lower  part  of 
the  meniscus  exactly  at  the  mark,  and  transfer  the  contents  to 
the  weighed  flask  allowing  to  drain  for  15  seconds  against  the 
inside  of  the  flask.  Weigh  again  to  within  an  accuracy  of  2  mg. 

1  c.c.  of  water  may  be  assumed  to  weigh  997  mg.     The  slight 
fluctuations  due  to  variations  in  room  temperature  may  be  neg- 
lected.   Calculate  the  correct  volume  of  the  pipet.     Clean  a  glass 
stoppered  buret  with  cleaning  fluid,  rinse,  fill  to  the  mark  with 
distilled  water,  empty  down  to  the  25  c.c.  mark  and  let  drain  for 

2  minutes.    Then  adjust  the  meniscus  exactly  to  the  25  c.c.  mark 
and  be  sure  that  the   sides   of   the  buret  above  the  water  are 
entirely  free  from  drops  of  adhering  water.     Now  run  in  20  c.c. 
from  the  calibrated  pipet.     Compare  the  reading  obtained  with 
the  correct  value.     If  the   divergence   seems   large   consult  the 
instructor.     Next  calibrate  the   5   c.c.   pipet  by  transferring  its 
contents  5  times  to  the  buret,  beginning  with  the  meniscus  in  the 
latter  at  the  25  c.c.  mark.     Record  each  reading  but  note  particu- 
larly where  the  fourth  comes.     If  the  5  c.c.  pipet  is  inaccurate 
make  a  new  temporary  mark  on  the  stern  and  repeat.     Inciden- 
tally record  the  value  of  each  5  c.c.  portion  of  the  buret.     The  J& 
10  c.c.  and  the  25  c.c.  pipets  can  also  be  calibrated  by  the  help^ 
of  the  buret. 

7 


For  calibrating  the  larger  pipets,  calibrate  first  I  dry  100  c.c. 
volumetric  flask  with  the  most  accurate  20  or  25  c.c.  pipet,  mak- 
ing if  necessary  a  new  temporary  mark  on  the  stem  of  the  flask. 
When  this  flask  is  again  dry  use  it  for  checking  up  .the  values 
of  a  50  or  a  100  c.c.  pipet.  The  volumetric  flasks  larger  than 
100  c.c.  need  not  be  calibrated.  If  a  flask  is  rinsed  with  a  little 
alcohol  and  left  to  drain  over  night  it  will  usually  be  found  to 
be  perfectly  dry  the  following  day. 

2.  Preparation  of  .5N  Oxalic  Acid  (500  c.c.).— The  usefulness 
of  oxalic  acid  as  a  starting  point  for  the  preparation  of  standard 
acids  and  alkalis  is  due  entirely  to  the  fact  that  it  can  be  obtained 
chemically  pure  and  in  condition  suitable  for  direct  weighing.  Oxalic 
acid  is,  however,  not  a  strong  enough  acid  to  titrate  well  with  all 
the  common  indicators,  and  it  is  therefore  not  serviceable  for  acidi- 
metric  titrations  in  general.  But  by  means  of  oxalic  acid  and  with 
phenolphthalein  as  indicator,  standard  solutions  of  a  strong  alkali, 
like  caustic  soda,  can  be  obtained,  and  by  means  of  the  latter  stand- 
ard solutions  of  the  stronger  mineral  acids  can  then  be  prepared. 
.  The  reason  w4iy  the  strong  acids  and  alkalis  give  more  accurate 
and  reliable  results  is  the  fact  that  the  salts  which  they  form  when 
neutralized  are  no^  appreciably  hydrolyzed  by  water  into  acid  and 
base,  as  are  the  corresponding  salts  of  the  weaker  acids  and  bases. 
The  zone  of  neutrality  to  different  indicators  is  therefore  more 
sharply  defined,  and  corresponds  more  nearly  to  the  point  repre- 
sented by  the  presence  of  exactly  equivalent  amounts  of  acid  and 
alkali. 

Weigh  accurately  (to  the  fourth  decimal)  a  small,  clean,  and 
dry  beaker  or  large  crucible.  Then  add  to  the  weights  on  the 
balance  pan  15.7560  g.,  and  add  oxalic  acid  to  the  vessel  on  the 
other  side  until  exact  equilibrium  is  reached.  Dissolve  in  dis- 
tilled water  this  oxalic  acid  without  the  loss  of  a  single  crystal. 
The  acid  dissolves  rather  slowly.  The  solution  is,  therefore,  best 
made  in  a  beaker  by  the  aid  of  gentle  heating  with  about  250 
c.c.  water.  Transfer  every  drop  of  the  solution  to  a  measuring 
flask  (500  c.c.),  carefully  rinsing  the  last  traces  from  the  beaker 
into  the  flask  by  means  of  successive  small  amounts  of  cold 
distilled  water.  Cool  the  flask  in  running  tap  water  until  the 
contents  of  the  flask  have  reached  the  room  temperature.  (If 
a  thermometer  is  used  it  must  be  rinsed  carefully  before  it  is 
removed  from  the  flask.)  Fill  up  with  water  until  the  lower  side 
of  the  "meniscus"  is  exactly  even  with  the  500  c,ct  mark.  Stop-. 

9 


per  the  flask,  and  invert  several  times  (30-40)  so  that  the  solu- 
tion is  thoroughly  mixed.  Transfer  to  a  clean,  dry  bottle;  label 
and  preserve. 

Using  a  strong  base  like  sodium  hydroxid  and  a  sensitive  indi- 
cator like  phenolphthalein  for  the  titration,  it  is  possible  to  obtain 
quite  reliable  and  accurate  results  with  oxalic  acid.  The  volu- 
metric determinations  involved  in  metabolism  studies  and  urine 
analysis  are,  however,  extensively  based  on  titrating  ammonia, 
which  is  a  very  weak  base.  Phenolphthalein,  because  of  its  high 
degree  of  sensitiveness  to  weak  acids  and  its  lack  of  sensitiveness 
to  weak  bases,  is  useless  in  titrations  of  ammonia.  The  oxalic 
acid  and  the  phenolphthalein  are  therefore  used  only  for  the  pur- 
pose of  securing  a  standard  alkali  solution. 

3.  Preparation  of  Standardized  Sodium  Hydroxid.— The  so- 
dium hydroxid  used  for  titrations  must  be  as  free  as  possible 
from  carbonates,  because  otherwise  the  solutions  will  not  have 
the  same  titrating  value  with  all  the  common  indicators.  Sodium 
hydroxid  absorbs  rapidly  carbonic  dioxid  from  the  atmosphere 
and  should  therefore  not  be  exposed  to  the  air  more  than  is  un- 
avoidable. As  the  carbonates  are  insoluble  in  very  strong  sodium 
hydroxid  solutions,  clear  saturated  solution  should  be  used  as 
starting  point  for  the  preparation  of  standard  solutions. 

Transfer  about  60  c.c.  of  clear  saturated  sodium  hydroxid 
solution  to  a  large  bottle  and  add  1,200  to  1,500  c.c.  of  water. 
To  determine  the  exact  value  of  this  solution  it  is  only  necessary 
to  find  out  how  much  of  it  is  required  for  the  neutralization  of 
a  known  volume  of  the  half  normal  oxalic  acid  solution. 

Rinse  the  20  c.c.  pipet  with  the  oxalic  acid  solution  and  then 
measure  20  c.c.  into  a  beaker  or  flask.  Add  two  drops  of  indi- 
cator (i  per  cent,  alcoholic  solution  of  phenolphthalein). 

Rinse  a  buret  with  the  alkali,  fill  it  and  cover  with  a  test  tube. 
After  carefully  adjusting  the  meniscus  of  the  solution  to  the 
zero  point,  run  it  into  the  oxalic  acid  solution  more  and  more 
cautiously  toward  the  end  until  finally  one  single  drop  produces 
a  definite  and  stable  end  point.  Note  the  volume  of  alkali  re- 
quired (within  0.05  c.c.).  Repeat  the  titration  until  two  succes- 
sive ones  give  exactly  the  same  value. 

From  the  titration  figure  obtained  calculate  the  normality  of 
the  solution  and  how  much  of  it  must  be  taken  for  the  prepara- 
tion of  i  liter  of  tenth  normal  alkali. 

As  a  check  on  the  work  determine  the  normality  of  an  un- 

11 


known  hydrochloric  acid  solution  (furnished),  using  as  indi- 
cator (a)  phenolphthalein,  (b)  alizarin  red  (2  drops  of  I  per 
cent,  aqueous  solution).  A  dated  and  signed  report  on  the 
unknown  should  be  handed  in  before  making  the  tenth  normal 
alkali.  Label  and  preserve  the  standardized  alkali  solution. 

4.  Standardized    Hydrochloric    Acid. — Concentrated     hydro- 
chloric acid  is  approximately  a  10  N  solution  of  HC1.     With  a 
cylinder  transfer  60  c.c.  of  strong  hydrochloric  acid  to  a  large 
bottle;  add  1,500  c.c.  of  water  and  shake  very  thoroughly.     It  is 
preferable  but  not  absolutely  necessary  to  let  the  shaken  solution 
stand  over  night  before  titrating. 

Titrate  this  acid  in  the  same  way  as  the  oxalic  acid  solution, 
but  using  only  alizarin  red  as  indicator.  Calculate  the  normality, 
and  how  much  of  it  must  be  taken  for  the  preparation  of  I  liter 
of  tenth  normal  acid.  Label  and  preserve. 

5.  Tenth  Normal  Acid  and  Alkali. — From  the  standardized 
solutions  of  acid  and  alkali  prepare  I  liter  of  tenth  normal  hydro- 
chloric acid  arid  I  liter  of  tenth. normal  alkali.     Titrate  the  acid 
so  prepared   (20  c.c.)   with  the  tenth  normal  alkali.     The  two 
should  be  equivalent.     Determine  the  normality  of  an  unknown 
acid  with  the  tenth  normal  alkali.     Hand  in  a  dated  and  signed 
report  giving  the  value  obtained  for  the  unknown  and  giving  also 
the  titration  figures  for  the  tenth  normal  acid. 

Label  and  preserve  the  tenth  normal  solutions.  The  two  alkali 
solutions  do  not  always  keep  their  value  unchanged  because  more 
or  less  alkali  is  given  off  by  the  glass  containers.  The  hydro- 
chloric acids  solutions  keep  indefinitely.  If  discrepancies  are 
found  later  between  the  acid  and  the  alkali,  the  acid  should  be 
taken  as  correct. 

6.  Strong  and  Weak  Acids;  the  Use  of  Different  Indicators. 
(A)    Titrate  25  c.c.   tenth   normal   hydrochloric   acid   with   the 
tenth  normal  alkali,  using  as  indicator   (a)   phenolphthalein   (b) 
methyl  orange    (c)    alizarin  red.     Repeat  the  above  mentioned 
three  titrations  in  the   presence   of    10  c.c.   ammonium   chlorid 
solution  (2  per  cent.).     Repeat  the  titration  with  each  indicator 
using  in  place  of  the  hydrochloric  acid  (a)  25  c.c.  .iN  phosphoric 
acid  (b)  25  c.c.  .iN  lactic  acid. 

Record  the  titrations  in  tabular  form : 

Phenolphthalein:      Methyl  orange:          Alizarin  red: 
c.c. — End  point.*     c.c. — End  point.*     c.c. — End  point.* 

*  Sharp,  fair  or  indeterminate. 

13 


HC1: 

HC1  NH4C1 : 
Phosphoric 

acid: 
Oxalic  acid : 

(B)  Dilute  10  c.c.  tenth  normal  hydrochloric  acid  to  100  c.c., 
making  an  approximately  o.oi  N  solution.  (Measuring  cylin- 
ders are  accurate  enough  for  the  dilutions  referred  to  here.) 

From  this  o.oi  N  solution  prepare  four  100  c.c.  portions  of 
more  dilute  acids,  viz. :  o.ooi  N ;  0.0004  N ;  o.oooi  N  ;  o.ooooi  N. 
Arrange  in  a  row  four  test  tubes,  as  nearly  as  possible  of 
the  same  size,  and  transfer  to  each  one  5  c.c.  of  one  of  the  four 
dilute  acid  solutions.  To  the  contents  of  each  tube  add  one  drop 
(no  more)  of  a  0.15  per  cent,  alcoholic  solution  of  tetrabromo- 
phenolsulfonephthalein  ("bromphenol  blue"),  and  compare  the 
colors.  The  approximate  hydrogen  concentrations  of  these  solu- 
tions are  as  follows : 

CH  pH* 

o.ooiN... io-3  3.0 

O.OOO4N 4xio-4  3.4 

o.oooi  N io-4  4.0 

o.ooooi  N 10-*  5.0 

Add  the  same  amount  of  indicator  to  (a)  5  c.c.  o.ooi  N  lactic 
acid,  (b)  5  c.c.  o.ooi  N  acetic  acid,  (c)  5  c.c.  o.ooi  M  mono- 
potassium  phosphate.  Determine  the  approximate  pH  of  each 
of  these  three  solutions  by  comparing  their  colors  with  the  dilute 
hydrochloric  acid  solutions.  Although  the  total  acid  concentra- 
tion is  the  same  in  the  o.ooi  N  solutions  of  hydrochloric,  lactic, 
and  acetic  acids,  and  monopotassium  phosphate  (an  acid  salt), 
the  hydrogen  ion  concentration  (and  therefore  the  degree  of 
dissociation)  is  obviously  different  in  each  case.  In  any  such 
series  of  acid  solutions  of  the  same  total  concentration  (o.ooi  N 
in  this  instance),  the  hydrogen  ion  concentration  is  less  (and  the 
pH  greater)  the  weaker  the  acid.  •  The  strength  of  an  acid  is 
measured  by  its  dissociation  constant  (k),  a  figure  approximately 
equal  to  the  hydrogen  ion  concentration  of  a  solution  of  the  acid 
that  has  been  just  half  neutralized.  The  dissociation  constants 
of  the  three  weak  acids  used  in  this  experiment  are  given  below, 

*The  hydrogen  exponent  (pa)  is  the  logarithm  of  the  hydrogen  ion 
concentration  (CH)  with  the  minus  sign  omitted, 

15 


along  with  those  of  other  acids  and  bases  of  biological  impor- 
tance. 

Hippuric  acid 2.2x10-4  Uric  acid 1.5x10-6 

Acetoacetic  acid   . .  .  1.5x10-4  Carbonic  acid   ....  3.0x10-7 

Lactic  acid 1.4x10-4  Primary   phosphate  2.0x10-7 

Acid  oxalate  ......  3.0x10-5  Boric  acid   6.6x10-10 

j3  -Hy  droxybutyric 

acid 2.0x10-5 

Acetic  acid 1.8x10-5  Ammonia  ...... .,. .  1.8x10-5 

1.  Acidity  of  Gastric  Contents.  — The  acidity  of  the  normal 
stomach  contents  is  due  almost  wholly  to  hydrochloric  acid.  In 
pure  gastric  juice,  the  concentration  of  hydrochloric  acid  is  about 
0.15  N,  but  the  acidity  of  the  material  usually  found  in  the 
stomach  is  less,  as  a  result  of  dilution  and  partial  neutralization. 
When,  under  abnormal  conditions,  the  concentration  of  hydro- 
chloric acid  becomes  very  low,  certain  microorganisms  are  able 
to  grow  in  the  stomach  contents,  producing  lactic  acid.  It  is 
nevertheless  an  easy  matter  to  distinguish  between  a  relatively 
low  concentration  of  hydrochloric  acid  and  a  relatively  high  con- 
centration of  lactic  acid,  since  the  latter  is  a  much  weaker  acid. 

To  5  c.c.  o.oi  N  hydrochloric  acid  in  a  test  tube  add  just  one 
drop  of  a  0.4  per  cent,  alcoholic  solution  of  thymolsulfonephtha- 
lein.  Add  the  same  amount  of  indicator  to  (a)  5  c.c.  o.ooi  N 
hydrochloric  acid,  and  (b)  5  c.c.  o.i  N  lactic  acid.  Compare  the 
colors.  The  hydrogen  ion  concentration  of  the  o.i  N  lactic  acid 
should  be  less  than  that  of  the  o.oi  N  hydrochloric  acid. 

8.     Colorimetric  Determination  of  Hydrogen  Ion  Concentration. 

— The  hydrogen  ion  concentration  of  most  biological  fluids  is 
considerably  less  than  in  the  solutions  tested  in  the  preceding 
experiments,  and  dilute  hydrochloric  acid  solutions  cannot  be 
used  here  as  standards,  owing  to  the  ease  with  which  the  pH  is 
changed  by  slight  contamination.  Instead,  it  is  necessary  to  have 
a  series  of  standard  buffer  mixtures,  whose  pH  is  not  readily 
altered. 

A  suitable  set  of  stock  solutions  from  which  to  prepare  such 
standards  is :  0.2  M  monopotassium  phosphate,  0.2  M  acetic  acid, 
0.2  M  boric  acid  (containing  also  0.2  M  potassium  chlorid),  and  0.2 
M  sodium  hydroxid.  The  sodium  hydroxid  solution  must  be  prac- 

17 


tically  free  from  carbonate,  and  should  not  contain  calcium  or  bari- 
um. The  compositions  of  the  standard  mixtures  (diluted  to  200  c.c. 
in  each  case)  are  given  in  the  table  below.  These  mixtures,  once 
made  up,  can  be  relied  upon  for  only  about  one  week,  but  the  stock 
solutions  from  which  they  are  prepared  should  keep  indefinitely  in 
receptacles  of  resistance  glass,  except  the  sodium  hydroxid  solution, 
which  will  gradually  increase  in  strength  unless  kept  in  a  paraffined 
bottle. 

The  determination  is  carried  out  as  follows:  With  a  pipet 
transfer  two  c.c.  of  the  unknown  solution  to  a  measuring  cylin- 
der and  add  water  to  make  the  total  volume  20  c.c.f  Mix,  and 
add  one  drop  (no  more)  of  phenol  red  solution.  Compare  the 
color  with  the  set  of  standards. f  In  case  the  color  is  beyond 
the  limits  for  phenol  red  on  either  side,  repeat  with  the  next 
indicator  in  order  (see  table),  until  the  unknown  has  been  cor- 
rectly matched  against  one  of  the  standards.  The  pH  reading 
may  be  made  to  one-tenth  unit  by  adding  or  subtracting  o.i  in 
case  the  color  lies  definitely  between  two  consecutive  standards. 

Determine,  in  the  manner  described,  the  hydrogen  ion  concen- 
tration of  two  unknowns  (supplied).  The  same  method  will 
later  be  applied  to  urine. 

Indicator:     METHYL  RED.*  ,' 

50  c.c.     0.2  M  CH3COOH  and  23.0  c.c.     0.2  M  NaOH          4.6 

50  c.c.  29.0  c.c.  4.8 

50  c.cj  34.5  c.c.  5.0 

50  c.c.  38.5  c.c.  5.2 

50  c.c.                      J  42.5  c.c.  5.4 

50  c.c.  45.0  c.c.  5.6 
*Dimethylaminoazobenzene-o-carboxylic   acid    (0.4  per   cent,   alcoholic 
solution;  use  one  drop). 

Indicator:     BROMCRESOL   PURPLE.f 

50  c.c.     o.2MKH2PO4         and    3.7  c.c.     0.2  M  NaOH  5.8 

50  c.c.                  fcft                           5-7  c.c.  6.0 

50  c.c.                     "                            8.6  c.c.  6.2 

50  c.c.                     "                          12.6  c.c.  6.4 

50  c.c.                     "                          17.8  c.c.  6.6 
tDibromo-o-cresolsulfonephthalein     (0.04    per    cent,    aqueous    solution 
of  monosodium  salt;  use  2  drops). 

*In  this  work,  pipets  should  never  be  blown  out,  and  water  should 
not  be  taken  from  a  wash  bottle  that  has  been  blown  into,  since  a  small 
amount  of  carbon  dioxid  readily  spoils  the  result. 

tA  series  of  diluted  standard  mixtures,  with  the  indicators  already 
added,  will  keep  for  a  short  time,  and  offers  the  most  convenient  ar- 
rangement when  a  great  many  pH  determinations  are  being  made  simul- 
taneously. 

10 


Indicator:     PHENOL  RED.* 

50  c.c.     o .  2  M  KH2PO4  and  23 . 6  c.c.  o .  2  M  NaOH          6 . 8 

50  c.c.  29.6  c.c.  7.0 

50  c.c.  35.0  c.c.  7.2 

50  c.c.  39.5  c.c.  7.4 

50  c.c.  42.8  c.c.  7.6 

50  c.c.  45.2  c.c.  "                7.8 

50  c.c.  46.8  c.c.  8.0 
*  Phenolsulfonephthalein    (0.02  per   cent,   aqueous    solution   of   mono- 
sodium  salt;  use  3  drops). 

Indicator:     THYMOL   BLUE.f 

50  c.c.     0.2  M  H3BO3,KC1    and    5.9  c.c.  0.2  M  NaOH  8.2 

50  c.c.  8.5  c.c.  8.4 

50  c.c.  12.0  c.c.  8.6 

50  c.c.  16.3  c.c.  8.8 

50  c.c.  21.3  c.c.  9.0 

t  Thymolsulfonephthalein  (0.04  per  cent  aqueous  solution  of  mono- 
sodium  salt;  use  2  drops). 

9.  Special  Test  for  Hydrochloric  Acid. — Gunzberg's  reagent  (2 
g.  phloroglucin  and  i  g.  vanillin  in  100  c.c.  alcohol)   is  very  re- 
liable as  a  means  of   distinguishing  between   hydrochloric  acid 
and  lactic  or  other  organic  acids.     The  reaction  is  best  carried 
out  as  follows : 

Transfer  5-6  drops  of  the  reagent  to  a  shallow  evaporating  dish, 
and  evaporate  to  dryness  over  a  water  bath  consisting  simply  of 
a  beaker  of  boiling  water.  The  alcoholic  solution  spreads  all  over 
the  dish,  leaving  a  thin  coating  of  the  dry  reagent.  By  means 
of  pipets,  or  glass  tubes  drawn  out  like  pipets,  transfer  one  drop 
of  .01  N  hydrochloric  acid  to  one  side  of  the  dish,  and  on  another 
side  deposit  one  drop  of  .1  N  lactic  acid,  and  again  place  the  dish 
on  the  water  bath.  A  purplish  ring  is  quickly  formed  around 
the  hydrochloric  acid  drop  while  the  lactic  acid  remains  colorless. 

Heating  over  the  flame  may  be  substituted  for  the  water  bath, 
but  the  least  overheating  tends  to  obscure  the  reaction  by  char- 
ring. This  reaction  is  extensively  used  in  the  examination  of 
stomach  contents. 

10.  Special  Test  for  Lactic  Acid. — More  or  less  specific  tests  for 
lactic  acid  are  known  and  are  considered  important  because  of 
the  frequency  with  which  lactic  acid  is  found  in  the  stomach  con- 
tents of  those  suffering  from  carcinoma  of  the  stomach.    A  con- 
venient yet  reliable  method  is  the  following: 

To  5-10  c.c.  of  .1  N  lactic  acid  (or  filtered  stomach  juice)  in 
a  large  test  tube  add  a  few  drops  (.5  c.c.)  of  normal  hydrochloric 
acid  and  about  10  c.c.  of  ether.  By  cautiously  inverting  the  test 

21  ' 


tube  during  3-4  minutes  (taking  care  to  avoid  explosions  due  to 
expanding  ether  vapors)  the  lactic  acid  is  in  part  taken  up  by  the 
ether.  By  means  of  a  25  c.c.  or  50  c.c.  pipet  and  suction,  remove 
the  lower  aqueous  layer  as  completely  as  possible.  Decant  the 
remaining  ether  into  another  test  tube  so  as  to  free  it  from  the 
few  drops  of  aqueous  solution  not  taken  out  by  the  pipet.  Then 
add  to  the  ether  solution  .2  per  cent,  ferric  chlorid  solution,* 
a  little  at  a  time  with  shaking,  until  the  maximum  yellow  color  is 
obtained.  The  amount  of  solution  added  and  the  depth  of  the 
color  obtained  give  a  rough  index  as  to  the  amount  of  lactic  acid 
present. 

Old,  deep  colored,  ferric  chlorid  solutions  frequently  fail  to 
give  the  test  for  lactic  acid.  By  the  addition  of  hydrochloric  acid, 
in  the  proportion  of  i  c.c.  concentrated  acid  to  5  c.c.  of  10  per 
cent,  ferric  chlorid  solution,  the  pale  color  of  the  latter  and  its 
value  as  a  reagent  for  lactic  acid  are  restored. 

i  c.c.  of  acidified  10  per  cent,  ferric  chlorid  solution  diluted 
with  40-50  c.c.  of  tap  water  gives  a  suitable  solution  for  the  test. 

11.  Nitrogen  Determination  in  Ammonium  Salts. — The  most 
convenient  and  useful  analysis  of  nitrogenous  products  of  phys- 
iological significance  is  the  determination  of  the  nitrogen.  The 
nitrogen  of  such  products  can  be  split  off  by  hydrolysis  in, the 
form  of  ammonia,  which  can  then  be  determined  by  distillation 
and  subsequent  titration. 

In  a  small  beaker  weigh  (to  the  fourth  decimal)  3-3.5  g.  pure 
ammonium  sulphate.  The  salt  contains  traces  of  water  (.5-1 
per  cent.),  unless  it  has  been  dried  by  heating  1-2  hours  at  about 
110°  C. ;  it  should  be  kept  in  a  desiccator  over  sulphuric  acid. 
Dissolve  the  salt  without  the  loss  of  a  single  crystal  in  a  500  c.c. 
volumetric  flask,  add  1-2  c.c.  concentrated  hydrochloric  acid,  and 
fill  up  to  the  mark  with  water.  The  acid  is  added  to  keep  ouf 
moulds.  Mix  thoroughly  and  transfer  to  a  dry  bottle,  or  to  a 
bottle  freshly  rinsed  twice  with  about  25  c.c.  of  the  solution. 

This  solution  should  be  stoppered,  labeled,  and  preserved  as  a 
standard  solution.  It  is  used  to  check  up  the  accuracy  of  ammonia 
determinations  and  sulphate  determinations,  and  later  for  colori- 
metric  nitrogen  determinations. 

*  10  c.c.  of  10  per  cent,  ferric  chlorid  solution  in  400-500  c.c.  of  tap 
water. 

23 


By  means  of  a  pipet  transfer  25  c.c.  of  the  ammonium  sul- 
phate solution  to  a  300  c.c.  Kjeldahl  flask.  Add  with  a  cylinder 
75  c.c.  of  water,  and  add  also  a  small  pinch  of  talcum  powder 
(to  prevent  bumping  during  the  boiling).  Put  the  flask  in  a 
clamp  so  that  the  bottom  is  about  I  cm.  above  the  top  of  a  micro 
burner. 

Transfer  25  c.c.  of  tenth  normal  hydrochloric  acid  to  a  300  c.c. 
Florence  flask;  add  water  enough  to  make  a  volume  of  about 
150  c.c.  and  add  two  or  three  drops  of  indicator  (alizarin  red). 

The  indicator  is  added  at  the  beginning  so  that  if  by  any  chance 
the  ammonia  distilled  over  is  more  than  enough  to  neutralize  the 
acid  that  fact  is  at  once  revealed.  When  this  happens  add  more 
standard  acid  (10  or  25  c.c.). 

Add  about  5  c.c.  of  saturated  sodium  hydroxid  solution  to  the 
contents  in  the  Kjeldahl  flask  and  connect  immediately  by  means 
of  a  rubber  stopper  and  glass  tubes  with  the  receiver  containing 
the  acid.  Light  the  burner  without  delay,  to  prevent  back  suc- 
tion, and  boil  vigorously  for  not  less  than  7  minutes,  counting 
from  the  time  the  boiling  begins.  Withdraw  the  receiver  so  that 
the  delivery  tube  is  well  above  the  liquid  before  removing  the 
flame.  Cool  the  receiver  in  running  tap  water.  Titrate  the  re- 
maining uncombined  acid  with  tenth  normal  alkali.  From  the 
figures  obtained  calculate  the  amount  of  nitrogen  recovered  (in 
milligrams)  and  compare  with  the  theoretical  figure  which  the 
amount  of  ammonium  sulphate  taken  should  give. 

In  calculating  the  nitrogen  from  the  titration  figures,  the  amount 
of  acid  combined  with  the  ammonia  can  be  regarded  as  a  tenth  nor- 
mal nitrogen  solution,  each  cubic  centimeter  of  which  accordingly 
represents  1.4,  or  more  accurately  1.401,  milligram  nitrogen.  Ex- 
ample: 25  c.c.  .1  N  HC1  was  the  original  amount  of  acid  in  the 
receiver.  After  the  distillation  the  titration  of  the  distillate  re- 
quired 2.1  c.c.  of  .1  N  NaOH.  The  ammonia  had  therefore  neutral- 
ized 25 — 2.1  or  22.9  c.c.  of  the  tenth  normal  acid,  22.9 Xi. 4  —  32.06 
(milligram  nitrogen). 


c.c.  volumetric  flask  dissolve   1-1.5  g.  accurately  weighed  urea.X 
Add  a  few  drops  (.5-1  c.c.)  concentrated  hydrochloric  acid,  a^d     x. 


12.     KjeldahFs  Method  for  Determining  Nitrogen. — In   a    100       \ 

a^d    N 

make  the  volume  up  to  100  c.c.,  mix,  and  transfer  to  a  clean  anchv 
dry  bottle,  label,  and  preserve. 

Pipet  5  c.c.  of  this  solution  into  each  of  two  Kjeldahl  flasks, 

25 


f/ 


Cu 


00  <se 


95-%?^ 


add  15  c.c.  concentrated  ammonia  free  sulphuric  acid  and  2  c.c. 
5  per  cent,  copper  sulphate  solution,  and  boil  30-40  minutes. 

When  organic  substances  are  boiled  with  strong  sulphuric  acid 
hnth  nyidatinn  and  hydrolysis  take  place.  The  oxidation  occurs 
at  the  expense  of  the  oxygen  in  the  sulphuric  acid,  and  the  latter  is 
consequently  reduced.  The  sulphurous  fumes-  thus  produced  are 
very  irritating  to  the  mucous  membranes  of  the  nose  and  throat. 
The  digestion  must,  therefore,  be  made  in  a  hood  having  a  reason- 
ably good  draft. 

Instead  of  a  hood  a  "fume  absorber"  can  be  used.  By  the  help 
of  an  ordinary  water  pump  (of  glass)  the  fumes  are  then  partly 
aspirated  directly  into  the  drain  pipes,  and  the  remainder  is  col- 
lected in  the  lower  part  of  the  fume  absorber. 

In  Kjeldahl's  method  sulphuric  acid  is  used  for  the  destructive 
digestion  but  other  substances  are  added  to  hasten  the  process. 
These  accessory  substances  act  either  as  catalyzers  (copper  sul- 
phate or  mercury)  or,  when  added  in  large  quantities,  they  raise 
the  temperature  and  thus  hasten  the  digestion.  Potassium  sul- 
phate (5-20  g.)  is  most  commonly  used  for  the  purpose  of  rais- 
ing the  temperature.  In  the  modification  here  described  a  mix- 
ture of  sulphuric  acid  (i  volume)  with  phosphoric  acid  (3  vol- 
umes) is  substituted  for  sulphuric  acid.  This  mixture  gives  a 
very  high  temperature,  but  it  acts  on  glass  much  more  rapidly 
than  sulphuric  acid  alone  and  can  not  therefore  be  used  except 
in  connection  with  digestions  which  can  be  completed  in  a  few 
minutes.  It  is  probably  the  best  for  the  destructive  digestion  of 
urine.  And  only  5  c.c.  are  taken  for  each  digestion  instead  of 
15-20  c.c./the  amount  required  in  the  case  of  sulphuric  acid. 

The  acid  mixture  is  prepared  as  follows:  To  50  c.c.  of  a  5 
per  cent,  copper  sulphate  solution  add  300  c.c.  of  85  per  cent, 
phosphoric  acid  and  mix.  Add  100  c.c.  of 'concentrated  sulphuric 
acid  (free  from  the  least  trace  of  ammonia),  mix,  and  cover 
well,  to  prevent  absorption  of  ammonia  from  the  air. 

A  10  per  cent,  solution  of  ferric  chlorid  is  also  required. 

When  urea  is  decomposed  by  means  of  boiling  concentrated 
sulphuric  acid,  it  is  simply  hydrolyzed  into  carbonic  acid  and 
ammonia  and  a  solution  of  ammonia  in  a  very  large  excess  of 
acid  is  obtained.  The  presence  of  all  this  acid  must  be  taken 
into  account  when  preparing  to  remove  the  ammonia  by  distilla- 
tion. (The  amount  of  alkali  required  should  be  determined  by  a 

27 


rough  titration  of  5  c.c.  of  the  acid  dissolved  in  500700  c.c.  of 
tap  water,  adding  the  saturated  alkali  with  a  measuring  cylinder.) 
A  20  to  30  per  cent,  excess  of  alkali  should  be  added  for  the 
distillation. 

With  a  pipet  transfer  5  c.c.  of  the  urea  solution  (or  urine)  to 
a  Kjeldahl  flask  (cap.  300  c.c.)  ;  add  5  c.c.  of  the  phosphoric  sul- 
phuric acid  mixture  and  2  c.c.  of  ferric  chlorid  solution ;  add 
also  3  or  4  pebbles,  to  prevent  bumping.  Fix  the  flask  in  a 
clamp  (in  a  hood)  so  that  the  bottom  is  only  about  I  cm.  above 
the  top  of  a  micro  burner.  Boil  with,  a  full  flame  until  all  the 
water  is  driven  off  and  the  flask  becomes  filled  with  white,  dense 
fumes.  At  this  point  cover  the  mouth  of  the  flask  with  a  watch 
glass  and  note  the  time.  Continue  the  heating  (without  chang- 
ing the  flame)  for  two  minutes.  At  the  end  of  two  minutes 
reduce  the  flame ;  the  white  fumes  should  now  be  confined  within 
the  flask.  With  the  small  flame  the  heating  is  continued  for  two 
minutes,  making  a  total  boiling  period  of  four  minutes,  counting 
from  the  time  the  mouth  of  the  flask  was  closed.  Remove  the 
flame  and  let  cool  for  not  less  than  four  nor  more  than  five 
minutes  and  then  add,  with  a  cylinder,  50  c.c.  of  water.  If  the 
cooling  process  is  made  too  long  the  contents  in  the  flask,  now 
chiefly  metaphosphoric  acid,  solidify  and  then  do  not  mix  well 
with  water. 

The  ammonia  is  to  be  distilled  into  a  receiver.  The  latter 
should  contain  from  25  to  75  c.c.  of  tenth  normal  acid,  the 
amount  depending  on  how  much  ammonia  is  expected.  The 
receiver  should  also  contain  water  enough  to  make  a  volume  of 
about  150  c.c.  Without  adding  more  water  to  the  hot  acid  solu- 
tion in  the  Kjeldahl  flask  introduce  the  necessary  alkali,  usually 
15  c.c.  of  saturated  sodium  hydroxid,  and  connect  promptly 
with  the  receiver.  With  the  bottom  of  the  flask  only  about  i  cm. 
from  the  top  of  the  micro  burner,  boil  vigorously  for  five  min- 
utes, counting  from  the  time  the  solution  begins  to  boil  hard. 
At  the  end  of  five  minutes,  withdraw  the  receiver,  allowing  it 
first  to  rinse  itself  with  steam  for  a  few  seconds.  Beginners  can 
advantageously  replace  the  receiver  with  another  flask  or  a  beaker 
containing  100  c.c.  of  water,  indicator,  and  a  drop  of  tenth  nor- 
mal acid,  and  continue  distillation  for  two  or  three  minutes,  so 
as  to  be  sure  that  none  of  the  ammonia  failed  to  get  into  the 
first  receiver. 

Cool  the  first  distillate  and  titrate  and  compare  the  nitrogen 

29 


value  obtained  with  the  theoretical  figure  which  the  urea  should 
give. 

The  process  for  the  determination  of  the  total  nitrogen  in 
urine  (5  c.c.)  is  exactly  the  same  as  the  process  described  above 
for  urea.  In  urine  there  is,  however,  considerable  complex 
organic  matter  to  be  oxidized  and  some  charring  and  foaming  is 
encountered.  With  urine  it  is  usually  necessary  to  have  not  less 
than  50  c.c.  of  acid  in  the  receiver  to  begin  with.  The  total 
nitrogen  is  likely  to  be  from  20  to  30  times  as  much  as  the 
ammonia  nitrogen  found  by  the  permutit  or  aeration  process. 

(P-  93-) 

13.  Determination  of  Nitrogen  in  Uric  Acid. —  Transfer  50-70 
mg.  of  pure  uric  acid  to  a  clean,  dry  test  tube.  Weigh  the  test 
tube  and  uric  acid  (to  the  fourth  decimal).  Shake  most  of  the 
uric  acid  into  a  dry  Kjeldahl  flask,  and  again  weigh  accurately 
the  empty  test  tube.  The  difference  between  the  two  weighings 
is  the  amount  of  uric  acid  taken.  In  the  same  way,  charge  an- 
other dry  Kjeldahl  flask  with  50-60  mg.  of  uric  acid. 

To  each  flask  add  5  c.c.  of  the  phosphoric  sulphuric  acid  mix- 
ture described  in  the  preceding  section.  Digest,  distil  and  titrate 
as  in  the  case  of  urea.  Calculate  the  absolute  and  percentage 
amount  of  nitrogen  and  compare  with  the  theoretical  figures. 


tART  II 
CATALYSIS,  CATALYZERS,  FERMENTS 

1.  Hydrogen-ion. —In  each  of  two  test  tubes  place  about  5  c.c. 
of  2  per  cent,  cane  sugar  solution.     To  one  add  5  c.c.  of  half 
normal  hydrochloric  acid  to  the  other  5  c.c.  of  water.    Heat  both 
in  a  beaker  of  boiling  water  for  ten  minutes.     Cool.     Transfer 
5  c.c.  of  a  sugar  reagent  (alkaline  copper  solution)  to  a  test  tube, 
add  about  one-half  c.c.  of  one  of  the  heated  cane  sugar  solutions 
and  boil  i  to  2  minutes.     Repeat  with  the  other  cane  sugar  solu- 
tion. 

Cane  sugar  when  split  by  hydrolysis  yields  reducing  sugars. 

2.  Hydroxyl-ion. — Fill  a  small  test  tube  up  to  within  about  I 
c.  from  the  top  with  i  per  cent,  tannic  acid  solution.    Add  a  few 
drops  sodic  hydrate  solution,  mix  quickly,  and  let  stand  for  a  few 
minutes. 

3.  Metallic  salts. — To  i  c.c.  of  urine  in  each  of  two  Kjeldahl 
flasks  add  5  c.c.  concentrated   sulphuric  acid.     To  one  add  a 
crystal  of  copper  sulphate.    Heat  to  gentle  boiling  for  10  minutes 
to  20  minutes.    Compare  the  rate  of  disappearance  of  the  brown 
color. 

4.  Pepsin.  — Prepare  a  pepsin  solution  from  the  mucous  mem- 
brane of  a  pig's  stomach  as  follows :    Strip  off  the  mucous  mem- 
brane from  a  pig's  stomach,  mix  with  300  c.c.  of  approximately 
decinormal  hydrochloric    acid    (the    "concentrated   hydrochloric 
acid"  is  approximately  a  10  N  solution)  in  a  wide-mouth  bottle 
(capacity  900-1,000  c.c.),  and  let  stand  over  night.     Remove  by 
decantation  75  c.c.  of  the  stomach  extract.     To  the  remaining 
mixture  in  the  bottle  add  5-10  c.c.  of  chloroform,  cork  tightly, 
shake  vigorously  for  a  few  seconds,  label,  and  place  in  an  incu- 
bator.   The  stomach  will  digest  itself  and  give  a  solution  suitable 
for  the  later  study  of  peptones. 

3.3 


Suspend  a  piece  of  egg  albumen,  or  a  Mett  tube,  in  the  top  of 
each  of  the  following  solutions : 

(a)  5  c.c.  of  undiluted  gastric  extract, 

(b)  5  c.c.  consisting  of  one  part  of  juice  to  3  parts  .1  N  hydro- 

chloric acid, 

(c)  5  c.c.  of  juice  diluted  as  in  (b)  and  heated  in  a  water  bath 

at  a  temperature  of  75°  for  15  minutes. 

Put  all  the  solutions  in  an  incubator  (the  warm  room)  over 
night.  Note  the  results  and  explain.  Compare  results  with  those 
of  other  students. 

To  20  c.c.  of  the  pepsin  solution  add  half  a  volume  of  disodic 
phosphate  solution  (10  per  cent.)  and  half  a  volume  of  calcium 
acetate  solution  (10  per  cent.).  Filter  off  the  precipitate  and 
wash  once  with  water.  Dissolve  the  precipitate  in  a  minimum 
quantity  of  half  normal  hydrochloric  acid.  Dialyze  over  night. 
Remove  the  liquid  from  the  dialyzing  tube,  test  its  reaction  with 
congo  red,  and  dilute  with  water  or  with  dilute  HC1  (which?) 
to  the  volume  of  the  pepsin  solution  originally  taken  (20  c.c.). 
With  this  solution  repeat  (a)  and  (b). 

5.  Trypsin. — Free  a  beef  pancreas  from  fat,  cut  up  fine,  and 
weigh ;  transfer  to  wide-mouth  bottle  and  add  3  c.c.  10  per  cent, 
alcohol  for  each  gram  of  pancreas.     Add  5  c.c.  of  chloroform, 
cork  tightly,  shake,  and  set  aside  for  two  or  three  days. 

Then  take  out  25-35  c-c-  of  tne  clear  liquid  and  pour  on  a 
filter.  Stopper  tightly  again,  and  put  the  bottle  in  the  warm  room 
(or  incubator)  to  be  preserved  for  later  experiments  on  "amino 
acids."  With  the  filtered  portion  of  the  pancreatic  extract  make 
the  following  experiment: 

Test  its  digestive  power  on  egg  albumen  (Mett's  tubes). 

6.  Urease. — i.  Heat  a  water  bath  from  50  to  55°  C.  Transfer 
35  c.c.  of  tenth  normal  hydrochloric  acid,  2  drops  of  alizarin  red, 
and  115  c.c.  of  water  to  a  300  c.c.  Florence  flask. 

Transfer  5  c.c.  of  2  per  cent,  urea  solution  to  a  300  c.c.  Kjel- 
dahl  flask.  Add  2  c.c.  of  neutral  phosphate  mixture  (o.2M  pri- 
mary phosphate  and  o.3M  secondary  phosphate;  pH  7.0).  Mix 
and  then  add  2  c.c.  of  5  per  cent,  alcoholic  Jack  bean  extract 
(p.  107)  ;  stopper  tightly;  place  the  flask  in  water  bath  at  50  to 
55°  and  shake  gently  for  i  or  2  minutes  to  promote  speedy  warm- 
ing. Continue  the  urease  digestion  for  exactly  10  minutes. 

35 


Cool  the  flask  to  promote  condensation  of  ammonia  vapors; 
then  add  50  c.c.  of  water,  5  to  7  drops  of  paraffin  oil  (to  prevent 
foaming)  and  2  g.  of  borax.  Connect  at  once  with  the  receiver, 
as  in  nitrogen  determinations;  distil  for  6  to  8  minutes.  Cool 
and  titrate. 

Calculate  how  much  of  the  urea  was  decomposed. 

2.  Repeat  the  experiment  described  under'  I  but  substitute  a 
water-bath  temperature  of  75-80°  C. 

3.  Repeat  at  room  temperature,  15  to  20°  C. 

4.  Repeat  at  room  temperature  but  substitute  2  c.c.  of  acid 
phosphate  (o.5M  primary  phosphate  and  o.oo6M  secondary  phos- 
phate; pH  5.0)  for  the  neutral  phosphate. 

5.  Repeat  with  2  c.c.  of  alkaline  phosphate  (O.OO4M  primary 
phosphate  and  O.5M  secondary,  phosphate;  pH  9.0). 

6.  Add  2   c.c.  of  mercuric  chlorid  solution  or  of  Nessler's 
reagent  to  a  previously  rinsed   Kjeldahl  flask.     Shake   for  one 
or  two  minutes.     Pour  out  the  mercury  solution  and  rinse  the 
flask  three  or  four  times  with  water.    With  the  apparently  clean 
flask  repeat  either  I  or  3.    Very  small  traces  of  mercury  destroy 
urease  as  well  as  many  other  enzymes. 

7.  Reversible  Reactions  (Mass  Law). — Mix  5  c.c.  methyl  ace- 
tate, 100  c.c.  water,  and  i  drop  concentrated  sulphuric  acid  in  a 
small  flask  (200-300  c.c.). 

(a)  Titrate  5  c.c. 

(b)  Boil  for  about  5  minutes,  using  reflux  condenser.    Cool, 
remove  5  c.c.,  titrate  the  acidity  (what  indicator?),  and  calculate 
the  acidity  for  100  c.c. 

Continue  the  boiling  for  one  hour,  and  repeat  the  titration. 

(c)  Mix  5  c.c.  of  glacial  acetic  acid  with  100  c.c.  of  methyl 
alcohol,  and  add  one  drop  of  concentrated  sulphuric  acid.     Re- 
move 5  c.c.,  dilute  this  with  water,  and  titrate  the  acidity.    Intro- 
duce the  preparation  into  a  250  c.c.  flask  attached  to  a  reflux 
condenser,  and  boil  for  two  hours.     Cool,  remove  5  c.c.,  dilute, 
and  titrate  as  before. 


37 


PART  III 
FATS 

1.  Solubility  of  Fats, — Test  the  solubility  of  tallow  in  water, 
5   per  cent.  NaOH,  ether,   chloroform,   and   alcohol,   carefully 
avoiding  the  vicinity  of  a  flame. 

Let  a  drop  of  the  ether  solution  fall  on  paper,  and  note  the 
result. 

Dissolve  in  3  c.c.  of  warm  benzene  enough  tallow  to  give  a 
moderate  precipitate  on  cooling.  Place  some  of  the  precipitate 
on  a  slide  under  a  cover  glass,  examine,  and  describe.  Note 
especially  the  shape  of  the  ends  of  the  individual  crystals. 

2.  "lodin  Number."    Degree  of  Unsaturation  of  Fats;  Wys' 
Method. — Start   simultaneously  the   determination   of  the  iodin 
number  of  cottonseed  oil  and  beef  tallow.    Weigh  about  .3  g.  of 
cottonseed  oil,  or  about  i  g.  of  beef  tallow,  into  a  250  c.c.  flask, 
and  dissolve  in  chloroform  (10  c.c.).     Add  25  c.c.  Wys'  iodin 
solution  with  a  pipet,  stopper,  and  put  in  a  dark  place  for  half 
an  hour.    Add  15  c.c.  of  10  per  cent,  potassium  iodid,  and  dilute 
with  100  c.c.  of  water,  titrate  the  excess  of  iodin  (partly  in  solu- 
tion in  the  water,  partly  in  the  chloroform)   with  .1  N  sodium 
thiosulphate,  by  running  the  latter  into  the  flask  until,  after  re- 
peated shaking,  both  the  chloroform  and  the  watery  solution  are 
but  faintly  straw  colored.    Then  add  a  few  drops  of  a  i  per  cent. 
starch  solution,  and  continue  the  titration  to  the  disappearance  of 
the  blue  color. 

While  waiting  for  absorption  to  take  place,  the  value  of  the 
iodin  solution  may  be  determined  in  terms  of  .1  N  thiosulphate 
by  adding  KI  and  titrating  in  the  same  way  as  above.  The 
difference  between  the  two  valves  represents  the  amount  of  iodin 
absorbed  by  the  fat,  and  is  calculated  in  grams  of  iodin  per  100  g. 
of  fat. 

Example : 

39 


•3  £•  cottonseed  oil,  when  treated  as  above,  required  35  c.c.  of 
.1  N  thiosulphate  for  ba^^titration. 

25  c.c.  of  the  iodin  solution  required  60  c.c.  of  .1  N  thiosulphate. 

The  oil  therefore  absorbed  iodin  corresponding  to  60  —  35  = 
25  c.c.  .1  N  thiosulphate,  i.e.,  25  c.c.  .1  N  iodin  or  25  X  .0127  g. 
Iodin  =  .317  g.  I.  The  Iodin  Number  is,  therefore,  -Vp  X  .31? 
=  105.6. 

WYS'  IODIN  SOLUTION.— Dissolve  13  g.  of  iodin  in  i  liter  of 
glacial  acetic  acid.  Titrate  the  iodin  content  of  the  solution,  and 
then  pass  washed  and  dried  chlorin  gas  into  the  solution  until  the 
titration  number  is  doubled.  A  very  distinct  change  in  the  color 
of  the  solution  indicates  when  this  has  taken  place. 

The  thiosulphate  solution  is  prepared  by  dissolving  24  g.  of  the 
crystallized  salt  in  i  liter  of  water  and  standardizing  it  in  the  usual 
way  (see  page  153). 

3.  Saponification  and  Preparation  of  Fatty  Acids. — Heat  about 
20  g.  of  beef  tallow  with  100  c.c.  of  alcoholic  solution  of  sodium 
hydrate  on  a  water  bath  over  night,  or  until  the  residue  is  dry. 
To  the  mixture  add  about  300  c.c.  water  and  heat  to  boiling. 
To  the  hot  solution  add  a  few  drops  of  methyl  orange;  while 
continuing  the  heating    (and  stirring),  acidify  with  dilute  sul- 
phuric acid,  and  filter.     Save  the  filtrate  which  contains  glycerin, 
then  wash  the  fatty  acid  residue  several  times  with  hot  water. 
Throw  away  the  washings. 

Transfer  the  "glycerol  filtrate"  to  an  evaporating  dish,  label, 
and  place  on  the  water  bath  for  evaporation  of  dryness. 

4.  Solubility  of  Fatty  Acids.— Test  the  solubility  of  the  fatty 
acid  mixture  prepared  from  tallow  in  water,  5  per  cent.  NaOH, 
ether,  alcohol,  and  benzol.     Compare  the  results  with  those  ob- 
tained with  fat. 

Let  a  drop  of  the  ether  solution  fall  on  paper,  and  note  the 
result. 

Dissolve  enough  of  the  fatty  acid  mixture  in  warm  alcohol  to 
give  moderate  precipitate  on  cooling.  Examine  the  crystals  under 
the  microscope,  and  describe  as  in  the  case  of  the  fat  crystals. 

5.  Spontaneous  Saponification  of  Fats.— Dissolve   about   .5   g. 
tallow,  or  a  few  drops  of  oil,  in  10  c.c.  warm  alcohol  in  a  test 
tube  (avoid  fire!).    Add  3-4  drops  phenolphthalein  solution,  and 

41 


. 

then,  drop  by  drop,  tenth  normal  sodic  hydrate  solution  (alcoholic 
sodic  hydrate  solution  is  best)  until  the  indicator  reveals  a  dis- 
tinctly alkaline  reaction.  Let  the  mixture  stand  in  a  warm  room 
over  night,  and  again  add  alkali  (drop  by  drop)  until  the  alkaline 
reaction  reappears. 

6.  Titration  of  Higher  Fatty  Acids. — Dissolve  about  .2  g.  fatty 
acid  mixture  in  10  c.c.   warm  alcohol  or  benzol    (avoid  fire!). 
Add  3-4  drops  phenolphthalein,  and  titrate  with  tenth  normal 
alcoholic  sodic  hydrate  solution  until  an  alkaline  reaction  is  ob- 
tained.    One  cubic  centimeter  of  the  alkali  corresponds  to  how 
much  fatty  acid  ? 

7.  Fat  digestion  with  Lipase  (Castor  Bean). —  Remove    the 
shells  from  10  g.  fresh  castor  beans,  break  them  up  as  fine  as 
possible,  and  allow  to  stand  over  night  in  a  loosely  stoppered 
test  tube  full  of  alcohol  ether  mixture.    Pour  off,  grind  the  beans 
to  a  powder  in  a  small .  mortar,  transfer  to  a  test  tube,  and  let 
stand  under  ether  over  night.    Filter  with  suction,  and  wash  two 
or  three  times  with  small  amounts  of  the  alcohol  ether  mixture. 
Grind  with  the  powder  in  the  order  named,  5  c.c.  .1  N  sulphuric 
acid  (supplied),  5  c.c.  of  neutral  cotton  oil   (Sp.  gr.  .92),  and 
5  c.c.  lukewarm  water.     The  water  should  be  added  a  little  at  a 
time  and  thoroughly  worked  into  the  mixture  so  that  at  the  end 
of  the  operation  a  good  emulsion  is  secured.     Cover  the  evapo- 
rating dish,  and  let  stand  in  a  warm  place  over  night. 

Add  50  c.c.  of  alcohol,  10  c.c.  of  ether,  and  a  few  drops  of 
phenolphthalein,  and  titrate  with  .5  N  sodium  hydrate.  Calculate 
the  amount  of  fatty  acid  and  the  per  cent,  of  fat  digested. 

8.  Glycerol  and  the  Acrolein  Test. — To  about  5  g.  acid  potas- 
sium sulphate  (KHSO4)    in  a  porcelain  crucible  add  one  drop 
of  glycerin,  heat  over  a  direct  flame,  and  note  the  pungent  odor 
and  tear-begetting  quality  of  the  fumes.     Note  how  much  heat 
must  be  applied  to  secure  an  unmistakable  test. 

When  the  filtrate  (saved  from  the  saponification  of  beef  tallow) 
has  evaporated  to  dryness,  the  residue  obtained  is  sodium  sul- 
phate ;  mixed  with  it  there  should  be  some  glycerin.  (How  much 
glycerin  might  be  there  ?) 

Mix  with  a  glass  rod  2-3  g.  of  this  residue  with  5-6  drops 
concentrated  sulphuric  acid  in  a  dry  crucible,  and  apply  heat.  If 

43 


an  unmistakable  acrolein  test  is  not  obtained,  repeat  with  more 
of  the  residue. 

9.  Emulsification. — Put  1-2  c.c.  of  a  solution  of  sodium  car- 
bonate (.2  per  cent.)  in  a  watch  glass,  and  place  in  the  center  a 
drop  of  rancid  oil.    The  oil  soon  shows  a  white  rim,  and  a  milky 
opacity  spreads  over  the  solution.    Note  with  the  microscope  the 
active  movements  in  the  vicinity  of  the  fat  drop,  due  to  the  sepa- 
ration of  minute  particles  of  oil. 

Examine  a  sample  of  milk  under  the  microscope.  The  fat 
should  be  in  a  state  of  fine  emulsion. 

10.  Lecithin. — Demonstrate  myeline  movements  (observed  on 
mixing  lecithin  with  water). 

Mix  a  small  piece  of  lecithin  in  water  in  a  test  tube.  Shake 
vigorously  for  a  time,  and  state  what  occurs.  To  the  contents 
add  concentrated  caustic  soda  and  boil.  Note  the  fishy  odor  of 
trimethylamine  (from  the  cholin).  Acidify  the  solution — fatty 
acids  are  precipitated,  and  glycerophosphoric  acid  is  left  in  solu- 
tion. Write  the  graphic  formula  for  lecithin. 

11.  Cholesterol;  Liebermann's  Reaction. — Dissolve  a  crystal  of 
cholesterin  in  10  c.c.  of  dry  chloroform,  and  to  this  solution  add 
a  few  drops  of  acetic  anhydrid    (formula?)    and  one   drop  of 
concentrated  H2SO4.    Shake.    The  liquid  becomes  rose  red,  blue, 
then  dark  green. 

12.  Problem. — On  the  basis  of  the  solubilities  and  reactions 
of  fats,  fatty  acids  and  soaps,  work  out  a  scheme  for  their  sepa- 
ration and  identification.     Apply  the  scheme  to  two  unknowns 
furnished.    Hand  in  a  dated  and  signed  report  on  the  same. 


45 


PART  IV 
CARBOHYDRATES 

The  numerous  very  old  methods  of  testing  for  sugar,  includ- 
ing Trommer's  and  Fehling's,  are  now  only  of  historical  interest, 
except  perhaps  in  connection  with  some  State  Board  Examina- 
tions. They  are  omitted  here. 

1.  Benedict's  Test.— One  of  the  best  qualitative  tests  for  sugar 
in  urine  by  means  of  copper  solutions  is  the  one  recently  pro- 
posed by  S.  R.  Benedict.    Benedict's  reagent  is  so  adjusted  that 
it  is  rather  more  sensitive  to  dextrose  than  Fehling's  solution, 
yet  is  not  reduced  by  creatinin  or  uric  acid,  and  little,  if  at  all, 
by  chloroform  (which  is  often  added  as  a  preservative  to  urine). 
Unlike  Fehling's  reagent  it  consists  of  a  single  solution.     The 
reagent  is  made  as  follows : 

Dissolve  85  g.  sodium  citrate  and  50  g.  anhydrous  sodic  car- 
bonate in  400  c.c.  of  water.  Dissolve  8.5  g.  copper  sulphate  in 
50  c.c.  of  hot  water.  Pour  the  copper  sulphate  solution  slowly, 
and  with  stirring,  into  the  alkaline  citrate  solution.  Filter  if 
necessary.  Label  and  preserve. 

Heat  to  boiling  about  5  c.c.  of  Benedict's  reagent  in  a  test  tube 
together  with  a  pebble  or  two,  to  prevent  bumping.  Add  about 
8  drops  of  sugar  solution  (or  urine)  and  boil  for  2  minutes.  If 
more  than  two-  or  three-tenths  per  cent,  of  sugar  is  present,  the 
solution  will  be  filled  with  a  colloidal  (greenish,  yellow,  or  red- 
dish) precipitate.  With  smaller  amounts  of  sugar  the  precipitate 
will  usually  appear  only  on  cooling  (the  cooling  should  not  be 
hastened  by  immersion  in  cold  water). 

2.  Folin-McEllroy's   Test   for   Sugar.— The    reagent    in    this 
test  is  made  as  follows :    Dissolve  100  g.  of  sodic  pyrophosphate, 
30  g.  of  disodic  phosphate  and  50  g.  of  dry  sodium  carbonate  in 
approximately  I  liter  of  water  by  the  aid  of  a  little  heat,    Dis- 

47 


solve  separately  13  g.  of  copper  sulphate  in  about  200  c.c.  of 
water.  Pour  the  copper  sulphate  solution  into  the  phosphate- 
carbonate  solution  and  shake. 

To  5  c.c.  of  the  solution  in  a  test  tube  add  5-8  drops  of  urine 
(never  add  more  than  0.5  c.c.)  and  boil  for  1-2  minutes  or  heat 
in  a  beaker  of  boiling  water  for  3  minutes.  If  more  than  the 
normal  traces  of  sugar  be  present  the  hot  solution  will  be  filled 
with  a  colloidal  (greenish-yellow  or  reddish)  precipitate  as  in 
Benedict's  test.  This  test  is  a  trifle  more  sensitive  than  Bene- 
dict's, therefore  when  working  with  urine  only  a  distinctly  posi- 
tive test  obtained  with  the  solution  still  hot  is  to  be  regarded  as 
positive. 

3.  Reduction  Test  for  Sugar  in  Normal  Urine.  —  That  most 
human  urines  contain  distinct  traces  of  reducing  sugar  can  be 
shown  by  the  use  of  more  sensitive  copper  reagents.  One  such 
reagent  can  be  made  as  follows: 

(A)  Dissolve  5  g.  of  crystallized  copper  sulphate  in  100  c.c. 
of  hot  water,  and  to  the  cooled  solution  add  60-70  c.c.  of  pure 
glycerin. 

(B)  Dissolve  125  g.  of  anhydrous  potassium  carbonate  (with 
stirring)  in  400  c.c.  water. 

Mix  one  volume  of  the  glycerin-copper  solution  (A)  with  two 
volumes  of  the  potassium  carbonate  solution  (B).  Only  small 
portions  should  be  mixed  at  a  time,  as  the  reagent  (after  mixing) 
does  not  keep,  but  undergoes  gradual  reduction. 

The  test  for  sugar  in  normal  urine  is  made  as  follows : 

First  transfer  5-10  c.c.  of  the  mixed  reagent  to  a  test  tube, 
and  boil  the  reagent  in  order  to  determine  the  extent,  if  any,  to 
which  the  reagent  is  spontaneously  reduced. 

Transfer  1-2  g.  picric  acid  to  a  small  bottle;  to  it  add  some  of 
the  urine  (10-50  c.c.)  to  be  tested,  insert  a  cork,  shake  for  five 
minutes,  and  filter.  By  this  treatment  the  disturbing  creatinin 
and  uric  acid  are  removed.  Now  heat  5~IQ  c-c-  °f  the  copper 
solution  to  boiling  in  a  fairly  wide  test  tube  (with  a  pebble  to 
prevent  bumping),  add  1-2  c.c.  of  the  filtered  urine,  and  boil  for 
60-75  seconds.  If  the  sugar  present  is  relatively  large  in  amount, 
the  solution  becomes  turbid  while  still  boiling,  as  in  Benedict's 
test.  If  no  such  turbidity  is  observed,  centrifuge  at  once,  i.e., 
before  cooling.  The  bottom  of  the  centrifuge  will  be  covered 

49 


with  cuprous  oxid.     The  crystals  to  be  observed  in  the  super- 
natant cooling  liquid  are  potassium  picrate. 

Repeat  this  test  with  1-2  c.c.  urine  which  has  not  been  treated 
with  picric  acid,  and  note  how  much  more  abundant  the  reduction 
is  with  ordinary  creatinin  containing  urine. 

4.  Phenylhydrazin  Test  (Osazone  Test). — To  5-10  c.c.  of  .2  per 
cent,  glucose  solution  in  a  test  tiifce  add  5  c.c.  of  a  phenylhydrazin 
solution  (containing  5  per  cent,  phenylhydrazin  hydrochlorid,  20 
per  cent,  sodic  acetate,  and  10  per  cent,  acetic  acid).    Heat  in  a 
beaker  of  boiling  water  for  half  an  hour.     Let  the  test  tube 
remain  in  the  beaker  until  the  water  has  cooled,  and  examine  the 
glucose  osazone  crystals  under  the  microscope. 

Write  the  reaction  involved  in  the  formation  of  osazone. 

5.  Glucose  Eeactions  versus  other  Carbohydrates.  —  Apply  one 
of  the  copper  reducing  tests,,,  and  the  phenlyhydrazin  test  to  .2 
per  cent,  solutions  of  arabinose,  levulose,  cane  sugar,  maltose, 
and  lactose. 

6.  Selivanoff's  Test  for  Ketose-Sugars.  —  SelivanofFs   reagent 
contains  .05  per  cent,  of  resorcin  and  about   12  per  cent,  of 
hydrochloric  acid. 

To  5-10  c.c.  of  the  reagent  in  a  test  tube  add  about  i  c.c.  of 
,i  per  cent,  solution  of  levulose,  boil  for  one  minute,  set  aside  to 
cool,  and  note  the  development  of  the  color. 

Repeat  the  test  with  dextrose  and  with  cane  sugar  (and,  if 
desirable,  with  other  dilute  sugar  or  carbohydrate  solutions). 
Tabulate  the  results. 

7.  Test  for  Ketose  Sugars  (Levulose?)  in  Urine.    —  Collect 
urine  for  one  hour  (preferably  just  before  the  noon  hour).    Then 
take  50  g.  of  cane  sugar  and  collect  urine  for  another  hour. 
Dilute  the  smaller  volume  of  urine  to  that  of  the  larger,  or  both 
to  a  convenient  small  volume.    To  10  c.c.  of  each  in  a  test  tube 
add  5  c.c.  of  10  per  cent,  lead  acetate  solution,  shake,  and  filter. 
Apply  Selivanoff's  levulose  test  to  about  2  c.c.  of  each  filtrate. 
Record  the  results  obtained. 

Minute  traces  of  levulose  in  human  urine  are  probably  not  so 
infrequent  an  occurrence  as  is  generally  assumed  to  be  the  case. 
The  color  obtained  with  urine  is,  however,  not  exactly  like  that 

51 


given  by  aqueous  levulose  solutions.  If  a  distinct  "levulose'  out- 
put from  the  cane  sugar  is  obtained,  test  both  urines  for  reducing 
sugar,  and  note  whether  there  appears  to  be  an  increase  of  sugar 
in  the  urine  of  the  second  period. 

8.  Orcin  Test  for  Pentoses. — The  pentose  reagent  is  made  by 
dissolving  i  g.  of  orcin  in  500  c.c.  30  per  cent,  hydrochloric  acid 
(6:1)  and  adding  i  c.c.  of  10  per  cent,  ferric  chlorid  solution. 

Heat  5  c.c.  of  the  orcin  solution  to  boiling  in  a  test  tube.  Re- 
move from  the  flame,  and  add  (immediately)  i  c.c.  i  per  cent, 
solution  of  arabinose. 

Repeat,  adding  i  c.c.  .2  per  cent,  arabinose  solution. 

Note  the  color  (violet,  blue,  red,  green)  and  the  formation  of  a 
precipitate. 

Repeat  with  I  per  cent,  arabinose  solution  previously  diluted 
(a)  with  4  volumes  of  urine  (b)  with  4  volumes  I  per  cent, 
glucose  solution. 

Repeat  (a)  with  urine  alone  (b)  with  i  per  cent,  glucose  solu- 
tion (c)  with  i  per  cent,  cane  sugar  solution. 

9.  Fermentation  Test  for  Sugar.  —  Certain  sugars  (which?) 
are  decomposed  by  yeast  into  carbon  dioxid  and  alcohol.     The 
formation   of   CO2  in   fermentation  has   been   extensively  used 
both  for  qualitative  studies  and  for  quantitative  determinations 
of  sugar.     Except  among  physicians  who  have  not  the  facilities 
for  making  other  tests,  the  fermentation  method  is  now  seldom 
used. 

To  some  .5  per  cent,  sugar  solution  (dextrose,  pentose,  cane 
sugar,  lactose)  in  a  test  tube  add  a  small  piece  of  yeast.  Shake 
to  make  uniform  mixtures,  and  with  each  fill  a  "fermentation 
tube."  Substitute  water  for  sugar  solution  in  a  control  test,  and 
set  the  tubes  aside  in  a  warm  place  over  night.  Record  the 
results. 

10.  Benedict's  Method  for  the  Determination  of  Sugar. — Pre- 
pare 500  c.c.  of  Benedict's  solution  as  follows:     Dissolve  9  g. 
pure  copper  sulphate  in  a  500  c.c.  volumetric  flask  with  about 
100  c.c.  distilled  water.    Dissolve  50  g.  anhydrous  sodic  carbonate, 
100  g.  sodium  citrate,  and  50  g.  sodium  sulphocyanate  in  250 
c.c.  distilled  water.     The  copper  sulphate  must  be  weighed  ac- 
curately on  the  analytical  balance.     Pour  the  copper  solution, 

53 


slowly,  with  stirring  and  without  loss  of  a  single  drop,  into  the 
alkaline  citrate  solution.  Then  pour  the  mixed  solution  back  into 
the  measuring  flask  without  loss,  add  5  c.c.  5  per  cent,  potassium 
ferrocyanid  solution,  and  with  the  rinsings  make  the  total  volume 
up  to  500  c.c.  Mix,  transfer  to  a  clean,  dry  bottle,  label,  and 
preserve.  Twenty-five  cubic  centimeters  of  the  solution  corre- 
sponds to  50  mg.  of  dextrose,  52  mg.  of  levulose,  67  of  lactose, 
or  74  of  maltose. 

The  determination  is  carried  out  as  follows : 

Measure  25  c.c.  of  Benedict's  solution  into  a  porcelain  dish,  add 
5-10  g.  of  solid  sodic  carbonate,  heat  to  boiling,  and  while  boiling 
run  in  the  sugar  solution  (or  urine)  fairly  rapidly  until  a  white 
precipitate  begins  to  form.  Then  add  the  solution  more  slowly 
(with  slower  boiling)  until  the  last  trace  of  the  blue  color  dis- 
appears. The  addition  of  the  sugar  solution  should  be  done  at 
such  a  speed  that  the  boiling  solution  is  kept  nearly  constant  in 
volume  during  the  operation.  The  original  sugar  solution  (or 
urine),  if  concentrated,  should  be  diluted  so  that  not  less  than 
10  c.c.  will  be  required  to  give  the  amount  of  sugar  which  the 
25  c.c.  of  reagent  is  capable  of  oxidizing. 

Five  divided  by  the  volume  of  sugar  solution  taken  gives  the 
per  cent,  of  sugar.  Check  the  value  of  the  reagent  by  determin- 
ing the  sugar  in  .5  per  cent,  dextrose  solution. 

11.  New  Method  for  Titration  of  Sugar.  —  (See  Journ.  Biol. 
Chem.,  33,  513,  1918;  38,  287,  1919.) 

1.  Alkaline  phosphate  mixture.     Powder  in  a  large  mortar 
200   g.    of    crystallized   disodic   phosphate    (HNa2PO41i2H2O), 
sprinkle  over  it  about  50  g.  of  sodium  thiocyanate  (or  60  g.  af 
potassium  thiocyanate).     Mix  with  mortar  and  spoon  for  about 
ten  minutes.    A  uniform  semi-liquid  paste  is  obtained.     To  this 
paste  add  120  g.  of  monohydrated  sodium  carbonate  (or  100  to 
no  g.  anhydrous  carbonate)   and  mix  with  mortar  and  spoon 
until  a  rather  fluffy  granular  powder  is  obtained.    Leave  in  mor- 
tar covered  with  paper  over  night,  then  mix  once  more.     This 
reagent  keeps  indefinitely  but  should  be  kept  in  stoppered  bottles 
so  as  not  to  lose  too  much  moisture. 

2.  A  saturated  solution  of  sodium  carbonate  containing  14 
to  20  per  cent.  Na2CO3. 

3.  A  copper  sulphate  solution  containing  59  g.  of  CuSO4, 
5H0O  and  2  c.c.  of  concentrated  sulphuric  acid  per  liter.     The 

55 


sulphuric  acid  in  this  reagent  is  added  only  to  prevent  precipita- 
tion of  copper  hydrate  by  the  traces  of  alkali  gradually  given  off 
by  glass.  The  solution  keeps  indefinitely. 

5  c.c.  of  the  copper  solution  is  reduced  by  25  mg.  of  glucose 
or  levulose,  by  40.4  mg.  of  anhydrous  lactose,  or  by  45  mg.  of 
anhydrous  maltose.  The  normal  reduction  time  is  5  minutes, 
except  in  the  case  of  levulose  which  is  reduced  within  2  minutes. 
In  working  with  lactose  it  should  be  noted  that  crystallized  lactose 
contains  I  molecule  of  water  of  crystallization. 

The  titration  is  best  made  in  large  hard  glass  test  tubes.  Urines 
or  sugar  solutions  up  to  a  concentration  of  7  or  8  per  cent,  are 
titrated  directly,  that  is  without  any  preliminary  dilution. 

A  special  sugar  buret,  total  capacity  5  c.c.,  divided  in  0.02  c.c. 
is  used  for  measuring.  This  buret  should  have  an  accessory  tip 
very  fine  and  about  5  cm.  long;  it  should  also  have  a  rubber  tube 
attachment  above  for  filling  by  suction. 

\Transfer  5  c.c.  of  the  copper  solution  to  the  large  test  tube; 
add  I  c.c.  of  sodic  carbonate  solution,  thereby  precipitating  the 
copper  and  rendering  the  solution  alkaline.  Add  5  g.  (not  less 
than  4.5  nor  more  than  5.5  g.)  of  the  solid  phosphate  mixture. 
Heat  gently,  with  shaking,  until  all  the  salts  except  for  a  few  iso- 
lated particles  of  sodium  carbonate  have  dissolved.  A  practically 
clear  solution  is  usually  obtained  in  less  than  i  minute  and  tem- 
perature need  not  excee'd  60°  C.  Use  only  a  micro  burner  as 
the  source  of  heat. 

From  the  sugar  buret,  filled  by  suction,  with  the  urine  or 
sugar  solution,  add  0.4  c.c.  to  i.o  c.c.  to  the  warm  clear  copper 
solution.  With  watch  in  hand  (or  clearly  visible)  heat  the  mix- 
ture rapidly  to  unmistakable  boiling.  Note,  on  the  second  hand, 
when  the  boiling  begins;  from  that  moment  keep  track  of  the 
time,  and  thereafter  heat  only  enough  to  keep  the  contents  just 
to  boiling — by  moving  the  test  tube  back  and  forth,  through  the 
flame.  When  bumping  begins  add  a  pebble  to  promote  ^ven, 
gentle  boiling. 

If  the  contents  of  the  test-tube  do  not  suddenly  become  turbid 
from  precipitated  cuprous  sulphocyanate  within  the  first  15  sec- 
onds of  boiling,  then  less  than  one-half  the  required  amount  of 
sugar  has  been  added  and  more  should  be  introduced  at  once. 
When  the  full  amount  of  sugar  (25  mg.)  is  present  the  turbidity 
appears  within  5  seconds  after  the  boiling  has  begun.  The  boil- 
ing should  normally  be  continued  for  3  minutes,  counting  from 

57 


the  time  that  the  boiling  point  was  reached,  before  any  more 
sugar  is  added.  Boil  I  minute  after  each  subsequent  addition 
of  sugar.  The  total  boiling  period  for  a  correct  titration  must 
not  be  less  than  4  or  more  than  6  to  7  minutes.  But  the  prelimi- 
nary titration  may  last  for  8  to  9  minutes  and  if  the  boiling  pro- 
cess has  been  gentle  the  result  will  then  be  only  about  I  per  cent, 
too  high. 

In  the  preliminary  titration  it  will  frequently  happen  that  the 
first  sugar  addition  contains  more  than  25  mg.  of  glucose,  and 
the  greater  the  excess  the  more  quickly  will  decolorization  of  the 
copper  take  place. 

Time  of  boiling  for  complete  reduction  of  copper  solution  by  an 
excess  of  glucose. 

Glucose  Boiling  Time 

Mg.  Min.  Sec. 

50  o  25 

40  o  40 

35  o  55 

30  I  20   tO    30 

27.5  i  30  to  55 

25-5  3 

By  noting  the  boiling  time  in  which  complete  reduction  has 
occurred  a  valuable  guide  to  the  amount  of  sugar  solution  to  be 
taken  for  the  next  titration  is  obtained. 

After  some  experience  has  been  gained  it  should  very  seldom 
be  necessary  to  make  more  than  two  titrations,  a  preliminary  and 
a  final,  for  any  one  sugar  determination. 

Calculation :  0.025  times  100,  or  2.5,  divided  by  the  titration 
figure  in  c.c.,  whether  this  be  several  c.c.  or  a  fraction  of  I  c.c., 
gives  the  per  cent,  of  glucose  present. 

Prepare  100  c.c.  of  i,  1.5  or  2  per  cent,  solution  of  pure  glu- 
cose and  learn  the  titration  on  the  basis  of  that  solution.  With- 
out some  preservative  moulds  are  apt  to  develop  in  this  solution 
in  the  course  of  3  or  4  days.  It  is  not  a  bad  plan  to  substitute 
tenth  normal  hydrochloric  acid  or  saturated  benzoic  acid  solu- 
tion for  one-half  of  the  water  used  in  making  the  solution.  Pre- 
servatives such  as  chloroform  or  toluol,  are  not  very  satisfactory 
in  this  case  because  they  contaminate  the  buret  so  that  drops  of 
sugar  solution  soon  begin  to  stick  to  the  sides. 

59 


12.     Polariscope  Method  for  the  Determination  of  Sugar.— The 

specific  rotation  of  a  substance  is  the  angle  through  which  the 
plane  of  polarized  light  is  turned  by  i  dm.  of  a  solution  containing 
i  g.  of  the  optically  active  substance  per  c.c. 

A  definite  temperature  and  light  of  a  definite  wave  length 
(sodium  light)  must  be  used  in  determining  specific  rotations. 

The  angle  of  rotation  -is  determined  by  means  of  some  form 
of  "polariscope"  (polarimeter,  saccharimeter,  etc.)  ;  and  the  spe- 
cific rotation  is  calculated  according  to  the  following  formula : 

Specific       Observed  Rotation  X  100 

Rotation = Percentage  X  Length  of  Observation  Tube  (dm.) 

If  the  specific  rotation  is  known,  as  in  the  case  of  the  common 
sugars,  the  per  cent,  of  sugar  is  obtained  by  the  following  trans- 
position of  the  above  formula : 

Observed  Rotation*  X  100 

Percentage  =  ^ ^ — ^  ,  A.  — r — r  ™  « — 71 — r 

Specific  Rotation  X  Length  of  Tube  (dm.) 

Following  are  the  specific  rotations  (yellow  light)  of  some  com- 
morP  suga«p : 

Glucose,  52.8 ;  Fructose,  — 93 ;  Cane  Sugar,  66.5 ;  Lactose,  55  ; 
Maltose,  137. 

The  determination  of  sugar  by  means  of  the  polariscope  is  as 
follows : 

Rinse  the  polariscope  tube  (length  usually  i  or  2  dm.)  with 
the  sugar  solution,  and  fill  almost  to  overflowing.  Place  the  glass 
plate  over  the  open  end  in  such  a  way  that  the  tube  does  not 
contain  any  air  bubble,  and  screw  on  the  cap.  Place  the  tube 
in  the  groove  of  the  polariscope.  Light  the  lamp,  and  move  the 
eyepiece  back  and  forth  until  the  lines  which  divide  the  field  are 
sharp.  Then  turn  the  screw  until  the  several  divisions  of  the 
field  are  equally  illuminated,  and  take  a  reading  by  means  of 
the  vernier.  The  circle  upon  the  disk  of  the  apparatus  is  di- 
vided into  quarter  degrees ;  24  divisions  of  the  vernier  correspond 
in  length  to  .25°.  Consequently  every  division  of  the  vernier 
corresponds  to  .01°.  Ascertain  whether  the  disk,  starting  from 
its  middle  point,  has  been  moved  to  the  right  or  to  the  left  of 
the  zero  point  of  the  vernier.  Read  off  the  number  of  whole 
degrees  and  hundredths.  Take  several  readings  by  moving  the 

61 


lever  and  coming  back  again  to  the  point  where  the  different 
parts  of  the  field  are  equal.  Correct  for  the  zero  point  of  the 
instrument  by  taking  readings  with  the  tube  filled  with  water. 
This  value  is  added  to,  or  subtracted  from,  the  reading  found 
with  the  sugar  solution. 

If  a  saccharimeter  is  used  instead  of  the  general  circular  polari- 
scope,  the  reading  on  the  scale  is  converted  into  angular  degrees 
of  rotation  by  multiplying  by  the  factor  .345.  The  percentage  of 
sugar  in  the  solution  is  then  calculated  by  using  the  formula 
given  above. 

13.  Cane  Sugar. — With  the  help  of  the  balance  prepare  100 
c.c.  of  a  known  cane  sugar  solution  (8  to  12  per  cent.)  So-called 
lump  sugar  is  better  for  this  purpose  than  granulated  sugar,  be- 
cause of  freedom  from  dust.  Determine  the  concentration  of 
the  solution  with  the  polariscope  according  to  the  directions  given 
in  the  preceding  section. 

Transform  a  part  of  the  solution  into  "invert  Sugar"  and  de- 
termine the  sugar  content  by  titration.  The  inversion  is  made  as 
follows : 

Transfer  10  c.c.  of  the  cane  sugar  solution  to  a  100  c.c.  volu- 
metric flask  and  add  i  c.c.  of  concentrated  hydrochloric  acid. 
Add  no  extra  water.  Heat  a  large  beaker  or  porcelain  dish  to 
70°  C.  When  the  water  has  reached  70°  C,  immerse  the  flask  in 
water  and,  without  allowing  the  temperature  of  the  water  bath 
to  sink  below  70°,  rotate  and  shake  the  flask  continuously,  but 
gently,  for  10  minutes.  At  the  end  of  this  time  cool  and  dilute 
to  the  100  c.c.  mark  with  water  and  mix. 

Instead  of  heating  to  70°  complete  inversion  can  also  be  accom- 
plished by  allowing  the  mixture  of  sugar  and  acid  to  stand  at 
room  temperatures  over  night.  The  over  night  process  is  of 
course  inapplicable  in  "practical  examinations." 

In  connection  with  titrations  of  invert  sugar  due  note  must 
be  taken  of  the  5  per  cent,  increase  in  the  weight  of  the  sugar 
accompanying  the  inversion.  In  titrating  unknown  solutions  of 
invert  sugar  it  must  not  be  forgotten  that  one-half  of  the  sugar 
is  levulose  and  that  levulose  reduces  alkaline  copper  solutions 
much  more  rapidly  than  does  dextrose.  Turbidity  within  the 
first  5  seconds  of  boiling  does  therefore  not  necessarily  indicate 
that  almost  enough  or  too  much  sugar  has  been  introduced. 

63 


14.  Problems.  — Determine  by  titration  and  by  the  polariscope 
the  sugar  concentration  of  one  unknown  solution  of  glucose,  one 
of  invert  sugar,  and  one  of  lactose.     Determine,  without  the  po- 
lariscope, the  glucose  and  cane  sugar  content  of  one  unknown 
mixture  of  these  two  sugars. 

Hand  in  dated,  signed  reports  on  these  unknowns ;  each  report 
to  represent  two  unknowns. 

15.  Preparation  of  Maltose. — Mix  10  g.  of  starch  with  30  c.c. 
of  cold  water  until  a  smooth  paste  is  obtained.    Pour  this  slowly, 
and  with  stirring,  into  250  c.c.  of  boiling  water  in  a  large  beaker, 
continue  the  boiling  1-2  minutes,  let  cool  to  75°  C,  stir  in  I  tea- 
spoonful  of  malt,  and  keep  at  this  temperature  until  the  mixture 
becomes  thin  and  watery.     Heat  again  to  boiling  with  stirring, 
cool  to  75°,  add  another  teaspoonful  of  ferment,  and  keep  at 
this  temperature  until  a  sample  no  longer  gives  any  color  with 
iodin  solution  (about  two  hours).     Boil,  cool,  transfer  to  a  250 
c.c.  volumetric  flask,  make  up  to  volume.    Determine  the  maltose 
by  titration  in  a  filtered  sample  and  calculate  the  amount  of  mal- 
tose obtained.    Taste  the  solution. 

16.  Preparation  of  Milk  Sugar. — To  300  c.c.  of  skimmed  milk 
diluted  with  800  c.c.  of  water,  add  cautiously  2  per  cent,  acetic 
acid  to  precipitate  the  casein.    When  enough  has  been  added  the 
liquid  is  nearly  clear.     Filter.    .Boil  the  nitrate,  and  filter  off  the 
coagulated  albumin.     Evaporate  the  nitrate  on  the  water  bath 
to  a  thin  syrup,  and  allow  it  to  stand  until  the  sugar  has  crystal- 
lized out. 

A  more  expensive  but  otherwise  much  better  method  for  pre- 
paring crystallized  lactose  from  milk  is  as  follows :  Transfer  100 
c.c.  of  skim  milk  to  a  200  c.c.  flask  or  bottle,  add  5  g.  of  solid 
picric  acid.  Shake  for  10  minutes,  let  stand  for  half  an  hour 
and  filter  into  a  300  c.c.  flask  or  bottle.  Add  to  the  filtrate  200 
c.c.  of  acetone  and  let  stand  for  24  or  better  for  48  hours.  Ex- 
amine and  test  the  crystalline  deposit.  It  is  all  milk  sugar. 

17.  Starch  and  Dextrin. — Heat  300  c.c.  of  water  to  boiling 
in  a  beaker.     Transfer  2  g.  of  finely  powdered  starch  to  a  test 
tube,  add  5  c.c.  of  cold  water,  shake  well  and  pour  the  resulting 
starch  suspension,  a  little  at  a  time,  into  the  boiling  water.    Boil 
gently  for  about  10  minutes.    Transfer  starch  solution  to  a  flask 
and  cool. 

65 


Prepare  also  about  50  c.c.  of  2  per  cent,  dextrin  solution,  using 
heat  if  necessary  to  get  a  clear  solution. 

Commercial  dextrin  sometimes  contains  unaltered  starch  and 
usually  contains  reducing  sugar.  If  appreciable  traces  of  starch 
are  present  the  gradual  addition  of  diluted  iodin  solution  gives 
at  one  stage  a  pure  blue  color.  If  no  starch  is  present  only  red, 
brown  or  violet  colors  appear. 

To  each  of  2  test  tubes  add  5  c.c.  of  water.  To  one  add  about 
2  c.c.  of  starch  solution  and  to  the  .other  about  2  c.c.  of  dextrin 
solution.  Add  diluted  iodin  solution  drop  by  drop  (by  means 
of  a  pipet)  to  each  test  tube  until  the  maximum  color  is  ob- 
tained. Note  the  color  obtained  in  each  case.  Heat  each  solution 
gradually  until  the  colors  have  disappeared  and  cool.  Heat  each 
test  tube  again,  this  time  to  distinct  boiling,  and  cool.  The  col- 
ored iodin  compounds  are  not  very  stable  at  higher  temperatures. 

Dilute  5  c.c.  of  the  starch  solution  to  100  c.c.  Add  varying 
amounts  of  this  diluted  starch  solution  to  2  c.c.  of  dextrin  so- 
lution and  apply  the  iodin  test.  Note  at  which  stage  of  iodin 
addition  that  the  pure  blue  color  is  obtained.  Note  also  that  a 
positive  starch  reaction  may  easily  be  missed  by  adding  too  much 
iodin.  The  iodin  solution  should  be  very  dilute;  the  ordinary  I 
per  cent,  solutions  need  be  diluted  20  times.  If  the  original  dex- 
trin solution  is  free  from  starch,  what  is  the  minimum  quantity 
of  added  starch  required  to  give  a  positive  pure  blue  reaction? 

To  5  c.c.  of  starch  solution  add  about  10  c.c.  of  saturated  am- 
monium sulphate  solution  and  shake.  Note  that  starch  is  pre- 
cipitated. Repeat  with  the  dextrin  solution,  if  the  dextrin  is 
free  from  starch  according  to  the  iodin  test,  it  will  not  give  a 
precipiate  with  ammonium  sulphate  solution.  The  starch  precipi- 
tation is  not  strictly  quantitative ;  a  faint  starch  reaction  can  usu- 
ally be  obtained  with  the  ammonium  sulphate  nitrate  from  starch 
solutions.  Try  this  (a)  with  starch  filtrates  (b)  with  mixtures 
of  starch  and  dextrin.  A  fairly  satisfactory  separation  of  starch 
from  dextrin  is  attained  by  the  help  of  ammonium  sulphate. 

Test  for  reducing  sugar  in  the  solutions  of  starch  and  dextrin. 

Mix  2  to  5  g.  of  brpwn  crackers  with  about  50  c.c.  of  cold 
water.  Filter.  Test  the  filtrate  for  starch,  dextrin,  and  reducing 
sugar. 

To  5  c.c.  of  saliva  in  a  small  flask  add  150  c.c.  of  the  starch 
solution.  (Fresh  starch  solutions  are  best  for  this  experiment.) 
Heat  in  a  beaker  of  warm  water  (40  to  42°  C),  Test  at  the  end 

67 


of  3  to  5  minutes  for  reducing  sugar  and  repeat  until  a  positive 
test  is  obtained. 

Continue  the  digestion  at  40  to  42°  C.  and  at  10  minute  inter- 
vals transfer  5  c.c.  portions  to  each  of  2  test  tubes.  To  one  add 
just  enough  iodin  to  give  an  unmistakable  color ;  to  the  other  add 
enough  iodin  to  give  the  maximum  color.  Save  the  series  of  test 
tubes  for  comparison  and  note  the  gradual  disappearance  of  the 
starch  and  the  formation  and  disappearance  of  dextrin. 

18.  Glycogen, — Cut  four  fresh  (Why  fresh?)  oysters  into 
small  pieces,  and  throw  into  four  times  their  weight  of  boiling 
water  slightly  acidulated  with  acetic  acid.  After  boiling  for  a 
short  time,  remove  the  pieces,  grind  in  a  mortar  with  some  sand, 
return  to  the  water,  and  continue  the  boiling  for  several  minutes. 
Filter  while  hot.  The  opalescent  solution  thus  obtained  is  an 
aqueous  solution  of  glycogen  and  other  substances. 

With  the  solution  of  glycogen  thus  obtained,  make  the  follow- 
ing tests : 

Add  iodin  solution  drop  by  drop  to  a  portion  of  the  glycogen 
solution.  The  liquid  will  assume  a  dark  red  color.  This  color 
disappears,  with  the  exception  of  the  color  due  to  the  iodin,  upon 
gentle  heating,  and  reappears  upon  cooling.  (Compare  with 
dextrin.) 

Test  the  glycogen  solution  with  Benedict's  solution  and  note 
the  result. 

Add  some  saliva  to  a  portion  of  the  glycogen  solution,  and 
put  in  the  warm  room  until  the  next  day.  Remove  and  divide 
into  two  portions.  Test  one  with  iodin  solution  for  glycogen, 
the  other  for  sugar.  Report  the  result. 


69 


& 


PARTY 
PROTEINS 

A  fundamentally  important  general  consideration  to  be  noted  in 
the  study  of  protein  materials  is  that  proteins  are  colloids,  and  that 
colloidal  solutions  differ  materially  in  their  physical  and  chemical 
reactions  from  the  corresponding  reactions  of  ordinary  true  solu- 
tions (of  crystalloids).* 

1.  Dialysis  of  Colloidal  and  Molecular  Solutions. — In    a    parch- 
ment  dialyzing  tube   place    15   c.c.   of  blood   serum    (protein)    and 
i   c.c.   of  strong  salt  solution,   Na2SC>4.     The  protein  is  present  in 
colloidal  form,  the  salts  in  molecular  (ionic)  solution.     Suspend  the 
tube  in  a  500  c.c.  beaker  of  distilled  water,  and  let  the  whole  stand 
over  night.     Test  a  portion  of  the  dialysate  for  protein  by  boiling  in 
a  test  tube  with  one  drop  of  dilute  acetic  acid.    Has  protein  passed 
through  the  membrane?     Test  the   dialysate   for  chlorids  and  sul- 
phates.    Do  salts  dialyze  through  parchment  membranes? 

2.  Suspension  Colloids;  Suspensoids.     PREPARATION. — Prepare   a 
colloidal  solution  of  gum  mastic  as  follows:     Drop  from  a  buret 
i  c.c.  of  saturated  alcoholic  solution  of  gum  mastic,  slowly  and  with 
stirring,  into  100  c.c.  of  distilled  water.    Filter. 

ACTION  OF  ELECTROLYTES. — To  10  c.c.  portions  of  the  above  filtrate 
add 

1.  5  c.c.  tenth  normal  HC1, 

2.  5  c.c.  tenth  normal  NaOH, 

3.  5  c.c.  normal  NaCl, 

4.  5  c.c.  urea  solution, 

5.  5  c.c.  concentrated  cane  sugar  solution. 
Let  stand,  note  what  occurs,  and  explain. 

*Fats  and  the  more  complex  carbohydrates  behave  also  as  colloids;  so 
indeed  do  all  substances  which  are  insoluble  in  water,  when  suspended 
in  water  in  a  state  of  sufficiently  fine  division.  Colloidal  solutions  of 
metallic  platinum  (one  of  the  least  soluble  of  metals)  are  frequently  em- 
ployed in  the  study  of  certain  ferment  reactions.  Proteins  occur  in  na- 
ture almost  wholly  in  the  form  of  colloidal  solutions. 

71 


EFFECT  OF  HEAT. — Boil  a  portion  of  the  solution  in  a  test  tube. 
Evaporate  10  c.c.  of  solution  to  dryness  on  the  water  bath,  and 
try  to  redissolve  it. 

3.  Emulsion  Colloids;  Emulsoids.— PREPARATION.— To   5   g.   gel- 
atin in  a  beaker  add  60  c.c.  of  water,  and  heat  gently  with  constant 
stirring  until  the  gelatin  is  all  dissolved. 

ACTION  OF  ELECTROLYTES. — To  10  c.c.  portions  of  the  gelatin 
solution  add, 

1.  A  few  drops  10  per  cent.  NaCl, 

2.  A  few  drops  concentrated  HC1. 

Evaporate  10  c.  c.  to  dryness  on  the  water  bath,  and  try  to  redis- 
solve. 

REVERSIBILITY  OF  COAGULATION  BY  ELECTROLYTES. — To  25  c.c.  of  a 
protein  solution  add  solid  ammonium  sulphate  with  shaking  until 
the  solution  is  saturated,  and  note  what  happens.  Filter  and  press 
the  precipitate  as  dry  as  possible  between  layers  of  filter  paper. 
Try  to  redissolve  the  precipitate  in  water. 

REVERSIBILITY  OF  COAGULATION  BY  HEAT. — To  another  25  c.c.  por- 
tion of  the  same  solution  add  2-3  drops. dilute  acetic  acid.  Heat  to 
boiling.  Filter,  press  the  precipitate  dry  as  before,  and  try  to  re- 
dissolve  in  water.  Tabulate  the  differences  between  emulsoids  and 
suspensoids. 

4.  "Hydrophile"  Colloids.*— To  some  gelatin  in  a  test  tube  add 
just  sufficient  water  to  cover  it.     Let  stand  and  note  changes. 

5.  Reversibility  of  "Sol"  and  "Gel"  States— Gelatinization  as 
a  Special  Case  of  Coagulation. — Warm  the  above  mixture  until  so- 
lution is  complete.     Cool  under  the  cold  water  tap.     What  occurs? 
Repeat  the  heating  and  cooling  a  number  of  times. 

6.  Coagulation  by  Oppositively  Charged  Colloids.— To   10   c.c. 
colloidal  arsenic  solution  add  10  c.c.  colloidal  Fe(OH)s.     Let  stand 
and  note  what  occurs. 

7.  Rate  of  Diffusion  of  Molecular  and  Colloidal  Solutions.— Melt 

some  4  per  cent,  gelatin  or  2  per  cent,  agar  jelly  in  a  beaker  by 
standing  in  hot  water.  Fill  5  small  test  tubes  ^  full  and  allow 
to  solidify.  To  the  jelly  tubes,  1-5  respectively,  add  an  equal  volume 
of 

1.  Colloidal  As2Ss, 

2.  Colloidal  Fe(OH)3, 

*  "Hydrophile"  colloids  are  one  class  of  emulsoids— not  all  emulsoids 
are  "hydrophilous." 

73 


3.  Picric  acid, 

4.  Copper  sulphate, 

5.  Congo  red. 

Allow  to  stand  2  days.  Compare  the  speed  of  diffusion,  i.e.,  the 
distance  which  the  various  substances  have  penetrated  the  jelly. 
What  is  the  relation  between  the  size  of  particles  and  rate  of  diffu- 
sion? 

1.  Test  for  Nitrogen,  Sulphur,  and  Phosphorus  in  Protein.— "Put 
a  little  dry  protein  *  into  a  dry,  cheap  test  tube.    Add  a  piece  of 
metallic  sodium  the  size  of  a  pea,  and  heat  strongly  for  a  few 
minutes.     Cool.     Carefully  and  without  handling  the  material, 
break  into  a  dry  evaporating  dish.     Cover  the  substance  with  a 
wet  filter  paper.    After  five  minutes  cautiously  add  25  c.c.  water. 
Stir  well.    Filter  into  a  tagypbe. 

(a)  To  5  c.c.  add  a  few  drops  of  ferrous  sulphate  and  a  drop 
of  ferric  chlorid  solution.    Warm  and  acidify  with  concentrated 
hydrochloric  acid,  noting  the  result. 

(b)  Acidify  5  c.c.  with  nitric  acid  and  add  a  few  drops  of 
ammonium  molybdate  solution.     Let  stand  over  night  and  look 
for  a  yellow  crystalline  precipitate. 

(c)  To  another  5  c.c.  add  a  few  drops  concentrated  sulphuric 
acid,  and  suspend  over  the  mouth  of  the  test  tube  a  piece  of  filter 
paper  previously  moistened  with  lead  acetate  solution. 

Discuss  the  action  of  the  sodium,  and  the  chemistry  of  the 
tests  (a),  (b),  (c). 

2.  Simple  Test  for  "Amid"  Nitrogen  and  for  Sulphur.  —  Put   a 

little  protein  in  a  test  tube  with  a  few  cubic  centimeters  of  strong 
sodium  hydrate  solution  and  three  drops  lead  acetate  solution. 
Heat  to  boiling,  and  suspend  a  piece  of  litmus  paper  over  the 
mouth  of  the  test  tube.  Explain. 

3.  Test  for  Phosphorus. — Boil  some  casein  in  the  hood  with 
10  c.c.   strong  nitric  acid  in   an   evaporating  dish.     Evaporate 
nearly  to  dryness,  add  25  c.c.  water,  and  test  for  phosphates. 

4.  Albumins. — Preparation  of  an  albumin  solution.  Consider- 
ing "egg  white"  to  contain   12  per  cent,  of  albumin,  prepare  a 
2   per   cent,   solution  by  suitable  dilution   with   distilled  water. 
Shake  thoroughly,  and  filter  through  a  plug  of  cotton. 

*  A  mixture  of  casein  and  dry  egg  albumen. 

75 


5.  Coagulation  by  Heating,— Heat  a  little  of  the  albumin  so- 
lution in  a  test  tube.    Compare  the  coagulation  so  obtained  with 
that  obtained  when  the  solution  is  diluted  (a)  20  times  with  dis- 
tilled water,  (b)  20  times  with  .1  per  cent,  salt  solution,  (c)  20 
times  with  .01  per  cent,  acetic  acid,    (d)   20  times  with  equal 
volumes  of  .1  per  cent,  salt  solution  and  .01  per  cent,  acetic  acid 

solution. 

* 

6.  Coagulation  Temperature. — Ascertain  the   temperature  of 
coagulation  of  albumin  as  follows :    Faintly  acidify  a'  portion  of 
the  solution  with  a  few  drops  of  .5  per  cent,  acetic  acid.    Filter 
if  necessary.    Place  a  portion  of  the  solution  in  a  test  tube,  insert 
a  thermometer  by  means  of  a  cork,  and  suspend  the  tube  in  a 
large  beaker  of  water.     Heat  the  beaker  slowly  with  a  small 
flame,  and  observe  the  point  at  which  the  albumin  solution  be- 
comes cloudy. 

7.  Sulphosalicylic  Acid  Test. — To  some  albumin  solution  in  a 
test  tube  add  a  few  drops  of  sulphosalicylic  acid  solution  (25  per 
cent?) .    Determine  the  delicacy  of  the  test. 

8.  Nitric  Acid  Test. — Put  5  c.c.  of 'the  solution  in  a  test  tube, 
and  introduce  5  c.c.  of  concentrated  nitric  acid  very  carefully 
with  a  pipet  to  the  bottom,  forming  an  under  layer.    Determine 
the  lowest  protein  concentration  at  which  the  test  is  unmistakable. 
Allow  10  minutes  if  the  reaction  is  slow  in  appearing. 

9.  Picric  Acid  Test. — Add  to  a  portion  of  the  albumin  solu- 
tion a  few  drops  of  a  solution  of  picric  acid  (i  per  cent.)  and 
citric  acid  (2  per  cent.) — Esbach's  reagent.    Determine  the  low- 
est protein  concentration  at  which  this  test  is  unmistakable. 

10.  Action  of  Ammonium  Sulphate. — Add   some  solid  ammo- 
nium sulphate  to  10  c.c.  of  the  albumin  solution  in  a  test  tube, 
shaking   frequently  until   the   solution   is   thoroughly  saturated. 
Allow  to  stand  for  a  while,  occasionally  shaking,  filter,  and  test 
the  filtrate  for  albumin  by  the  heat  test.     Test  the  solubility  in 
water  of  the  precipitate  on  the  filter  paper, 

11.  Action  of  Magnesium  Sulphate. — Perform   a   similar   ex- 
periment, using  solid  magnesium  sulphate  instead  of  ammonium 

77 


sulphate.    To  a  portion  of  the  filtrate  add  one  or  two  drops  acetic 
acid. 

12.  Biuret  Test— To  a  portion  of  the  albumin  solution  add 
a  little  sodium  hydrate,  then,  drop  by  drop,  very  dilute  copper 
sulphate.     The  solution  becomes  violet.     Study  the  delicacy  of 
the  reaction.     After  adding  to  the  albumin  solution  some  solid 
ammonium  sulphate,  repeat  the  test  with  (a)  the  same  amount 
of  alkali  (b)  a  large  amount  of  40  per  cent,  alkali. 

13.  Millon's  Test. — To  a  portion  of  the  albumin  solution  add  a 
few  drops  of  Millon's  reagent.     A  precipitate  forms,  which,  on 
heating,  becomes  brick  red.     Repeat,  using  a  dilute  solution  of 
phenol  instead  of  albumin.  .  On  what  group  in  the  protein  mole- 
cule does  this  test  depend?    Add  sodium  chlorid  and  repeat  the 
test.    Explain. 

14.  Xanthoproteic    Test.— To  a  few  c.c.  of  the  solution  add 
one-third  of  its  volume  of  concentrated  nitric  acid ;  a  white  pre- 
cipitate may  or  may  not  be  produced  (according  to  the  concentra- 
tion and  the  nature  of  the  protein).     Boil.     The  precipitate  or 
liquid  turns  yellow.     Allow  the  solution  to  cool,  and  add  an 
excess  of  ammonia.    Explain. 

15.  The  Glyoxylic  Acid  Reaction  (Hopkins  and  Cole). —  Treat 
2  or  3  c.c.  of  the  solution  with  the  same  volume  of  "reduced 
oxalic  acid."    Mix  and  add  an  equal  volume  of  concentrated  sul- 
phuric acid,  pouring  down  the  side  of  the  tube.     A  purple  ring 
forms  at  the  junction  of  the  fluids.     Mix  the  fluids  by  shaking 
the  tube  gently  from  side  to  side.     The  purple  color  spreads 
through  the  whole  fluid.     Repeat  in  the  presence  of  nitrates, 
chlorates,  nitrites,  excess  of  chlorids  and  carbohydrates,  respec- 
tively. 

"Reduced  oxalic  acid"  is  prepared  by  Benedict's  method  as  follows : 

To  10  g.  powdered  magnesium  in  a  flask  add  a  little  water,  and 

then  add  slowly,  with  shaking  and  cooling,  250  c.c.  of  cold  saturated 

oxalic  acid  solution.     Filter,  acidify  the  nitrate  with  acetic  acid,  and 

dilute  to  one  liter. 

16.    Acetic  Acid  and  Potassic  Ferrocyanid.— Acidify  some  al- 
bumin solution  in  a  test  tube  with  acetic  acid,  and  add  a  few  drops 

79 


of  potassic  ferrocyanid  solution.     A  white  flocculent  precipitate 
is  formed.    Determine  the  delicacy  of  this  precipitation. 

?  17.  Alcohol. — Add  an  excess  of  alcohol  (one  or  two  volumes) 
to  some  albumin  solution.  If  the  precipitate  is  small,  add  a  little 
dilute  sodium  chlorid  solution. 

18.  Tannic  Acid.— Make  some  protein  solution  slightly  acid 
with  .1  per  cent,  of  acetic  acid,  and  add  a  few  drops  of  tannic 
acid  solution. 

,    19.     Phosphotungstic  Acid. — Make  a  protein  solution  acid  with 
dilute  hydrochloric  acid,  and  add  a  few  drops  of  the  reagent. 

Globulins. — The  tests  are  made  upon  blood  serum. 

20.  Action  of  Carbon  Dioxid.— Dilute   5   c.c.   of  clear  serum 
with  45  c.c.  of  ice-cold  water.    Place  the  mixture  in  a  cylinder  or 
large  test  tube,  and  pass  through  it  a  stream  of  carbon  dioxid. 
What  is  the  effect  of  too  much  carbon  dioxid? 

21.  Precipitation  by  Dialysis. —Pour  20  c.c.  of  serum  into  a 
parchment  dialyzing  tube  previously  soaked  in  distilled  water. 
SuspenRhe  tube,  with  its  contents,  in  a  large  volume  of  water. 
Explain  the  precipitation. 

Pour  serum  drop  by  drop  into  a  large  volume  of  distilled  water 
(in  a  beaker).    What  takes  place?    Explain. 

22.  Precipitation  by  Magnesium  Sulphate.— Saturate  about  5 
c.c.  of  the  serum  with  magnesium  sulphate.    A  heavy  precipitate 
will  be  formed.     Compare  this  with  the  action  of  the  same  salt 
on  the  egg-albumen  solution. 

23.  Precipitation  with  Ammonium  Sulphate. — To    30   c.c.    of 
serum  add  an  equal  volume  of  a  saturated  solution  of  ammonium 
sulphate,  thus  obtaining  a  half-saturated  solution.     Filter  off  the 
precipitate,  wash  two  or  three  times  with  a  half-saturated  ammo- 
nium sulphate  solution,  and  dissolve  in  about  60  c.c.  of  water. 
This  yields  a  clear  solution  of  paraglobulin.     Apply  5  protein 
tests  to  this  solution. 

81 


&    24.     Keratin. — Show  that  keratin  (hair  or  horn)  is  a  protein. 

25.  Gelatin.— Make  dilute  gelatin  solution,  and  with  it  make 
6  tests  for  protein  (including  Millon's).    Test  for  sulphur. 

26.  Phosphoproteins. — Test  the  solubility  of  casein  in  water, 
dilute  acid,  dilute  alkali,  and  dilute  salt  solution. 

Make  six  protein  tests  on  a  solution  of  casein.  How  would 
you  test  for  albumin  and  casein  when  both  are  present?  Apply 
to  milk  and  to  unknown  furnished. 

27.  Peptones  (Proteoses).— For  the  following  experiments  use 
the  peptic  digestion  mixture  obtained  with  the  pig  stomach  (p. 
33).     Filter,  carefully  neutralize,  heat  to  boiling   (why?),  and 
again  filter.    Use  the  filtrate. 

To  a  small  portion  add  2  or  3  drops  of  dilute  acetic  acid  and 
a  few  c.c.  of  saturated  sodium  chlorid  solution.  Study  the  effect 
of  heating  and  cooling  on  this  precipitate. 

Apply  protein  tests  described  under  "albumin,"  and  record  the 
results  obtained. 

Dialyze  about  10  c.c.  of  the  peptone  solution  against  about  100 
c.c.  of  distilled  water  in  a  beaker.  After  24  hours  test  the  out- 
side water  for  peptones.  Explain. 

28.  Amino-acids,  Tyrosin  and  Leucin.  —  For  this  experiment 
use  the  pancreatic  digestion  mixture  prepared  for  the  study  of 
ferment  reactions  (p.  35). 

With  a  pipet  take  out  10  c.c.  of  clear  supernatant  liquid.  Filter 
this  portion  if  necessary;  dilute  it  with  2.  volumes  of  water,  and 
by  means  of  Mett's  tubes  determine  whether  the  proteolytic  fer- 
ment has  been  destroyed  or  is  still  active. 

Pour  the  rest  of  the  digestion  mixture  without  filtering  into  a 
good-sized  beaker  or  flask.  With  continuous  stirring  or  gentle 
shaking  to  prevent  burning  and  bumping,  heat  the  digestion  mix- 
ture until  it  begins  to  boil.  Some  care  is  needed  in  this  opera- 
tion because  of  the  presence  of  alcohol.  When  the  mixture  is 
boiling  remove  the  flame.  Note  approximately  the  volume  of  the 
mixture,  and  measure  into  a  test  tube  some  "Merck's  dialyzed 
iron,"  8-10  c.c.  for  each  100  c.c.  of  digestion  mixture.  Dump 
the  collodial  iron  into  the  digestion  mixture,  and  shake  or  stir 
vigorously.  Filter. 

83 


1 


V 

;ar, 


To  the  filtrate  add  a  few  drops  of  ammonia  and  5-10  g. 
bone-black.     Boil  for  a  few  minutes  and  again  filter.    A  clear 
faintly-colored  solution  should  be  obtained. 

Pour  this  last  filtrate  into  an  evaporating  dish,  acidify  with  a 
little  acetic  acid,  and  boil  down  to  about  one-sixth  of  the  original 
volume. 

Transfer  the  concentrated  liquid  to  a  flask  or  beaker,  and  set 
aside  in  a  cool  place  for  a  day  or  two.  Tyrosin  and  leucin  crys- 
tallize out,  the  former  first  and  in  much  greater  abundance. 
Examine  the  sediment  under  the  microscope. 

The  isolation  of  other  amino-acids  from  the  mother  liquor  iSv 
much  more  difficult. 

29.  Preparation  of  Cystin  (from  Wool). — Heat  50  g.  of  wool 
in  a  500  c.c.  flask  with  100  c.c.  concentrated  hydrochloric  acid 
on  a  water  bath  until  dissolved.  A  3-foot  glass  tube^should  be 
inserted  to  prevent  the%>ss  of  too  much  acid  liquid.  When  dis- 
solved boil  very  gently  o^r  a  small  flame  for  3-4  hours.  Add 
solid  sodjc  acetate  (100-130  g.)  until  no  free  mineral  acid  can  be 
detected  in  the  solution  by  means  of  congo  red  paper.  Allow  the 
mixture  to  stand  for  3-5  days.  The  longer  the  mixture  is  allowed 
to  stand,  up  to  3  weeks,  the  more  cystin  is  obtained.  Filter  on  a 
Buchner  funnel  and  wash  with  cold  water.  Then  dissolve  the 
precipitate  in  water  (150  c.c.) 'plus  5-10  c.c.  concentrated  hydro- 
chloric acid,  add  about  20  g.  purified  bone-black,  and  boil  5-10 
minutes. 

To  prepare  pure  bone-black,  let  the  impure  sample  stand  in  an 
excess  of  dilute  hydrochloric  acid  over  night,  filter,  and  wash  with 
cold  water  until  the  filtrate  is  neutral. 

Filter  again  with  suction,  heat  the  filtrate  to  boiling,  and  neu- 
tralize the  hot  hydrochloric  acid  by  adding  very  slowly  hot  con- 
centrated sodic  acetate  solution  (avoid  an  excess,  test  with  congo 
red  paper).  The  precipitate  formed  consists  of  cystin,  and  should 
be  very  white  and  pure.  If  it  is  dark  colored,  re-dissolve  in 
water  and  a  little  hydrochloric  acid,  and  repeat  the  bone-black 
treatment. 

Keep  the  mixture  boiling,  and  add  very  slowly  the  hot  sodic 
acetate  solution  until  the  crystallization  begins;  keep  hot,  and 
after  a  few  minutes  add  cautiously  a  little  more  acetate.  Well- 
formed  large,  and  characteristic  crystals  should  be  obtained. 

85 


Examine  the  crystals  under  the  microscope. 

Test  for  sulphur.  Test  also  for  ty rosin.  (Ordinarily  the 
crystals  are  quite  free  from  tyrosin.) 

Tyrosin  can  be  prepared  from  the  original  mother  liquor  by 
decolorizing  with  bone-black  and  letting  it  stand  in  a  cold  place. 


87 


PART  VI 
URINE  ANALYSIS  AND  METABOLISM 

Quantitative  urine  analysis  has  no  value  except  in  connection 
with  known  volumes  of  urine  representing  a  definite  known 
metabolism  period.  Even  in  student  exercises  involving  the 
learning  of  methods,  only  urines  which  represent  a  definite 
metabolism  period  should  be  used,  and  from  the  analytical  figures 
obtained  the  total  value  for  the  whole  urine  (and  metabolism 
period)  should  be  calculated. 

The  standard  common  metabolism  period  is  twenty-four  hours. 
The  only  correct  way  to  collect  twenty-four  hour  urines  is  to 
begin  the  metabolism  period  immediately  after  passing  the  night 
urine  in  morning.  Note  the  time  and  then  collect  all  the  urine 
passed  up  to  the  same  hour  the  following  morning.  The  reason 
for  this  rule  is  that  during  the  night  much  of  the  waste  products 
corresponding  to  the  food  intake  of  the  preceding  day  is  passed 
and  in  the  early  moaning,  before  any  food  has  been  taken,  the 
excretion  is  at  its  lowest  level. 

Sometimes  it  is  impracticable  to  collect  twenty-four  hour 
urines,  and  sometimes  it  is  desirable  to  study  the  urine  repre- 
senting shorter  metabolism  periods  such  as  three,  four  or  six 
hours.  Formerly  it  was  not  practicable  to  make  use  of  such 
short  periods,  because  the  analytical  procedures  required  too 
much  urine;  the  uric  acid  determination  alone  required  150  c.c. 
By  the  help  of  the  modern  colorimetric  methods,  nearly  com- 
plete analysis  can  be  made  on  the  basis  of  three  hour  urines. 
For  such  short  metabolism  periods  it  is  necessary  to  drink  not 
less  than  200  c.c.  of  water  at  the  beginning  of  the  period.  The 
period,  whether  consisting  of  three  hours  or  twenty-four  hours, 
should  begin  immediately  after  passing  the  night  urine. 

It  is  necessary  to  use  some  preservative  for  the  urine  unless 
the  analysis  can  be  completed  within  twenty-four  hours.  Chloro- 
form (2  c.c.)  or  toluol  or  xylol  should  be  introduced  into  the 

89 


empty  bottle  in  which  the  urine  is  to  be  collected  so  as  to  ex- 
clude from  the  beginning  any  possibility  of  bacterial  decompo- 
sition. 

In  systematic  urine  analysis  some  determinations  should  be 
made  as  soon  as  possible  (within  twenty-four  hours)  while 
others  can  be  postponed  as  long  as  desirable.  The  uric  acid  de- 
termination should  be  made  the  first  day,  because  the  uric  acid 
may  either  fall  out  as  a  sediment  or  may  be  decomposed  as  the 
result  of  long  standing.  The  creatinin  determination  should  be 
made  within  forty-eight  hours,  because  in  some  urines  it  is  grad- 
ually in  part  converted  into  creatin.  From  the  standpoint  of 
spontaneous  decomposition  in  well  preserved  urine  the  ammonia 
determination  can  be  postponed  indefinitely,  but  it  is  usually 
better  not  to  delay  the  ammonia  determination  more  than  two  or 
three  days.  In  turbid  urines  there  is  more  or  less  danger  of 
precipitation  of  a  part  of  the  ammonia  as  ammonium  magnesium 
phosphate. 

The  acidity  titration  should  be  made  within  twenty-four  hours. 
Most  urines  darken  very  much  on  standing  and  the  deepened 
color  makes  it  difficult  to  see  the  faint  color  which  marks  the 
correct  endpoint  of  the  titration. 

Standard  figures  for  the  composition  of  normal  twenty-four 
urines  are  abundant  in  the  literature.  (See  American  Journal  of 
Physiology  13,  45-115,  1905.)  Standard  figures  for  three-hour 
urines  are  as  yet  a  scarcity.  The  figures  recorded  below  for  the 
first  three-hour  morning  urines  were  obtained  in  1918  by  S. 
Youngburg.  The  subjects  were  medical  students. 

1.  Aeration  Method  for  the  Determination  of  Ammonia. — 
Measure  25  c.c.  of  the  ammonium  sulphate  solution  previously 
used  for  nitrogen  determinations  (p.  23)  into  a  tall  aerometer 
cylinder.  The  cylinder  is  fitted  with  a  two-hole  rubber  stopper 
and  glass  connections  so  arranged  that  compressed  (outside)  air 
(or  laboratory  air  previously  freed  from  ammonia)  is  passed  to 
the  bottom  of  the  cylinder  and  out  through  a  calcium  chlorid 
tube  filled  with  dry  cotton,  and  then  through  a  special  absorption 
tube,  into  a  receiver  containing  water  and  a  known  amount  of 
acid.  The  cotton  serves  the  purpose  of  holding  back  traces  of 
solid  sodic  carbonate,  formed  by  evaporation  on  the  sides  of  the 
cylinder. 

Add  to  the  ammonium  sulphate  solution  about  10  g.  sodium 

91 


COMPOSITION   OF   THREE-HOUR  MORNING  URINE. 


No. 

Total 
N 
gm. 

NH3-N 
mg. 

Urea-N 
gm. 

Uric 
Acid 
mg. 

Crea- 
tinin 
mg. 

Body 
wt. 
K. 

i 

.87   * 

42 

.72 

73 

176 

58 

2 

.00 

47 

•85 

66 

192 

56 

•3 

.02 

68 

.82 

56 

176 

59 

4 

.02 

79 

.82 

57 

224 

66 

5 

.12 

43 

•94 

48 

154 

60 

6 

•H 

74 

•97 

72 

163 

64 

7 

.18 

5i 

1.  00 

7i 

192 

66 

8 

•23 

107 

•99 

88 

204 

64 

9 

.26 

IOO 

.02 

85 

240 

80 

10 

.26 

4i 

.04 

65 

222 

79 

ii 

•33 

79 

.08 

64 

220 

77 

12 

•37 

77 

.16 

65 

190 

70 

13 

.42 

46 

•13 

88 

203 

54 

H 

.46 

88 

•23 

9i 

183 

67 

15 

.48 

58 

.29 

88 

229 

65 

16 

•51 

40 

.27 

69 

190 

64 

17 

•54 

34 

•35 

77 

212 

58 

18 

•55 

81 

•34 

67 

1  86 

61 

19 

20 

.88 
2.18 

69 
49 

.66 

•85 

89 
105 

247 
236 

11 

COMPOSITION    OF    THREE-HOUR    MORNING    URINE 


No. 

Volume 
c.c. 

Acidity 
in  c.c. 
o.iN 

H,P04 

mg. 

Total 
S 
mg. 

Inorganic 
S 
mg. 

Ethereal 
S 
mg. 

Cl 

g- 

i 

52 

34 

159 

46 

30 

4 

1.64 

2 

96 

6 

142 

54 

3i 

7 

i.  08 

3 

207 

30 

293 

H3 

39 

28 

i.  ii 

4 

61 

38 

247 

69 

5i 

4 

•54 

5 

204 

5 

12 

5i 

40 

0 

.90 

6 

81 

24 

86 

62 

38 

2 

1.  00 

7 

in 

40 

317 

96 

73 

6 

i.i5 

8 

185 

52 

38i 

99 

74 

7 

1.74 

9 

90 

44 

374 

107 

77 

5 

.62 

10 

138 

6 

19 

86 

50 

4 

i.i.S 

ii 

84 

35 

232 

86 

60 

ii 

•75 

12 

66  ' 

48 

369 

114 

93 

6 

.27 

13 

203 

26 

241 

76 

57 

4 

I.  21 

14 

1  02 

42 

283 

107 

38 

8 

I.  00 

15 

142 

6 

193 

95 

59 

22 

1.28 

16 

107 

33 

142 

105 

59 

24 

.76 

17 

5H 

13 

92 

78 

56 

10 

1.64 

18 

745 

3 

165 

105 

81 

7 

2.62 

19 

520 

17 

237 

65 

.47 

i 

i-57 

20 

139 

10 

261 

142 

93 

6 

1.29 

93 


chlorid,  about  2  g.  sodic  carbonate,  and  a  few  drops  of  kerosene. 
Do  not  add  any  water ;  the  greater  the  volume  the  longer  it  takes 
to  drive  off  all  the  ammonia.  Pass  a  very  strong  air  current 
through  the  mixture  for  one  and  one-half  hours,  and  collect  the 
ammonia,  which  the  air  carries  off,  in  a  receiver  containing  25 
c.c.  .1  N  acid  and  about  200  c.c.  water.  Titrate,  and  compare  the 
result  with  the  figures  obtained  by  distillation  (p.  25).  If  the 
results  are  too  low,  the  air  current  has  been  too  slow,  or  the  aera- 
tion process  has  not  been  continued  long  enough  to  drive  off  all 
the  ammonia. 

In  a  similar  manner  determine  the  ammonia  in  urine  (25  c.c.) 
and  calculate  the  24-hour  amount.  When  working  with  urine  it  is 
desirable,  though  not  absolutely  necessary,  to  substitute  10  g.  of 
potassium  oxalate  for  the  10  g.  of  sodium  chlorid.  Salts  hasten 
the  removal  of  the  ammonia,  and  oxalate  incidentally  prevents 
the  (possible)  formation  of  insoluble  ammonium,  magnesium 
phosphate. 

Save  the  remainder  of  the  ammonium  sulphate  solution  for 
sulphate  determinations. 

2.  Colorimetric  Method  for  the  Determination  of  Ammonia,  — 
With  an  Ostwald  pipet  measure  i  or  2  c.c.  of  the  ammonium 
sulphate  solution  into  a  large  Jena  test  tube  (200  x  25  mm.). 
Choose  the  amount  which  contains  nearer  i  mg.  of  nitrogen.  Fit 
the  test  tube  with  a  two-hole  rubber  stopper  carrying  an  inlet 
tube,  reaching  to  the  bottom,  and  an  outlet  tube.  Connect  the 
former  with  the  compressed  air  jet,  and  the  latter  with  an  ab- 
sorption tube  having  small  holes  drilled  through  the  wall  at  the 
end.  Insert  the  absorption  tube  into  a  100  c.c.  measuring  flask 
containing  20-30  c.c.  of  distilled  water  and  2  c.c.  .1  N  HC1.  Add 
2  drops  kerosene  and  a  few  drops  of  a  solution  containing  po- 
tassium oxalate  and  potassium  carbonate  (15  per  cent,  of  each), 
quickly  put  the  stopper  firmly  in  the  tube,  and  start  the  air  cur- 
rent, gradually  increasing  its  speed  for  about  two  minutes.  In 
ten  minutes  all  the  ammonia  should  have  been  driven  over  into 
the  receiving  flask. 

Remove  the  absorption  tube,  rinsing  it  with  water,  and  dilute 
the  contents  in  the  flask  to  about  75  c.c. 

Pipet  10  c.c.  of  standard  ammonium  sulphate  (p.  103)  into 
another  100  c.c.  measuring  flask,  and  dilute  with  water  to  60  c.c. 

Nesslerize  both  solutions  according  to  the  directions  given  in 

95 


the  alternative  colorimetric  method  (see  below)  but  using  only 
10  c.c.  of  Nessler's  solution  and  omitting  the  addition  of  sodium 
hydroxid. 

Make  the  color  comparison  according  to  the  directions  given 
in  the  alternative  method  (see  below). 

3.  Alternative  Colorimetric  Method  for  Determination  of  Am- 
monia.*— In  this  method  the  ammonia  is  extracted  from  the  urine 
by  gentle  shaking  with  a  synthetic  aluminate  silicate  powder  sold 
under  the  trade  name — permutit  (by  the  Permutit  Company, 
New  York).  Only  such  preparations  as  have  passed 
60  mesh  sieve  and  does  not  pass  through  an  .8o-«re?ri  sieve  should 
be  used.  Powders  of  any  desired  decree  of  fineness  are  obtain- 
able. 

Before  applying  this  method  to  urine,  use  it  for  the  determina- 
tion of  ammonia  in  the  ammonium  sulphate  employed  for  nitro- 
gen determinations  and  in  the  preceding  aeration  process.  Com- 
pare the  results  obtained  by  permutit  with  those  obtained  (a)iby 
distillation  (p.  25)  ;  (b)  by  the  marco  aeration  process  (p.  95). 

The  essential  mechanical  feature  of  this  new  reagent  for  ab- 
sorbing  ammonia  is  that  it  is  a  clean,  moderately  fine,  insoluble 
powder  which  gives  off  very  little  dust  or  turbid  material  to 
water,  and  settles,  like  sea  sand,  from  water  in  the  course  of  a 
few  seconds.  By  virtue  of  this  novel  feature  the  (absorbed) 
ammonia  can  be  separated  by  decantation  from  the  solution  (or 
urine)  which  contained  it. 

The  removal  of  ammonia  by  this  mineral  reagent  is  not  an 
absorptive  phenomenon.  The  reagent  is  a  complex  insoluble  so- 
dium salt  containing  active,  i.  e.,  easily  replaceable,  sodium,  and 
the  absorption  of  ammonia  involves  the  replacement  of  a  part  of 
this  sodium  by  ammonia.  The  chemical  affinity  of  the  active 
group  in  the  reagent  for  ammonia  is  remarkably  strong  so  that 
under  suitable  conditions  the  exchange  becomes  quantitative  as 
far  as  the  ammonia  is  concerned. 

While  the  chemical  reaction  involved  in  the  absorption  of  am- 
monia by  this  reagent  is  apparently  a  reaction  between  a  solid 
and  a  solution,  it  remains  to  be  said  that  the  solid  powder  con- 
tains about  20  per  cent,  of  water,  and  if  this  water  of  hydration 
is  removed  by  heat  the  activity  of  the  reagent  is  lost.  Even 
gentle  dry  heat  (100°  C.)  greatly  reduces  its  activity,  so  that  a 

*J.  Biol.  Chem.  XXIX.  329.  1917. 

97 


freshly  purified  and  rapidly  dried  product  is  less  active  than  the 
same  product  allowed  to  dry  at  ordinary  temperatures,  or  than 
the  same  product  dried  rapidly  at  100°  C,  and  allowed  to 
"weather"  for  a  day  or  two. 

An  important  characteristic  of  this  reagent  for  the  absorption 
of  ammonia  is  that  it  does  not  appreciably  deteriorate  by  being 
used.  After  washing  away  the  Nesslerized  ammonia  and  surplus 
alkali  first  with  water,  then  with  one  portion  of  2  per  cent,  acetic 
acid,  then  once  more  with  water,  the  powder  remaining  is  just 
as  efficient  as  before  for  the  absorption  of  more  ammonia. 

The  process  for  the  colorimetric  determination  of  ammonia  in 
urine  by  the  help  of  the  synthetic  zeolite  powder  is  as  follows: 

Transfer  about  2  gm.  of  the  powder  to  a  200  c.c.  volumetric 
flask.  Add  about  5  c.c.  of  water  (no  more),  and  with  an  Ost- 
wald  pipette  introduce  i  or  2  c.c.  of  urine,  or  with  a  5  c.c. 
pipette  introduce  5  c.c.  of  previously  diluted  urine  (corre- 
sponding to  I  or  2  c.c.  of  the  original  urine).  With  urines 
extraordinarily  poor  in  ammonia  it  may  be  necessary  to  use  more 
urine  (5  c.c.),  but,  in  so  far  as  it  is  practicable,  it  is  better  not 
to  use  more  than  2  c.c.  and  to  employ  a  weaker  standard  (0.5  mg. 
of  ammonia  nitrogen)  for  the  color  comparison.  Our  reason  for 
not  wishing  to  use  more  than  2  c.c.  of  urine  is  based  partly  on 
practical  experience  and  partly  on  the  recognition  of  the  fact 
that  the  salts  in  the  urine  tend  to  prevent  the  ammonia  absorp- 
tion from  being  quantitative.  Rinse  down  the  added  urine  by 
means  of  a  little  water  (i  to  5  c.c.),  and  shake  gently  but  con- 
tinuously for  5  minutes.  Rinse  the  powder  to  the  bottom  of  the 
flask  by  the  addition  of  water  (25  to  40  c.c.)  and  decant.  Add 
water  once  more  and  decant.  (In  the  case  of  urines  rich  in  bile 
it  is  advisable  to  wash  once  or  twice  more).  Add  a  little  water 
to  the  powder,  introduce  2  c.c.  of  10  per  cent,  sodium  hydroxid, 
shake  for  a  few  moments  and  set  aside,  while  preparing  the 
standard  ammonium  sulphate  solution  as  follows: 

Transfer  10  c.c.  of  the  standard  ammonium  sulphate  solution 
(p.  103)  containing  I  mg.  of  nitrogen  to  another  200  c.c.  volu- 
metric flask  and  add  2  c.c.  of  10  per  cent,  sodic  hydrate  (to  bal- 
ance the  alkali  added  to  the  permutit  mixture  in  the  other  flask). 
Dilute  to  about  150  c.c.  and  mix.  Transfer  20  c.c.  of  Nessler's 
solution  (see  p.  203)  to  a  measuring  cylinder.  Now  give  the 
volumetric  flask  a  vigorous  whirl  so  as  to  set  the  solution  spin- 
ning within  the  flask  and  add  at  once  the  whole  of  the  Nessler 

99 


solution  in  the  cylinder.  With  another  whirling  mdvement  se- 
cure the  complete  mixing  of  the  contents  in  the  flask.  If  the 
process  of  Nesslerization  has  been  successful  a  deep  red  but 
crystal  clear  solution  is  obtained.  If  it  is  not  perfectly  clear 
throw  it  away  and  prepare  a  fresh  standard.  With  a  little  experi- 
ence no  trouble  is  encountered  in  getting  clear  solutions.  When 
the  standard  solution  is  thus  satisfactorily  Nesslerized,  dilute  the 
contents  in  the  flask  containing  the  permutit  and  the  urinary 
ammonia  to  about  150  c.c.,  whirl  the  mixture  and  add  the  Nessler 
reagent  (20  c.c.)  exactly  as  in  the  case  of  the  standard  solution. 
Dilute  the  contents  of  both  flasks  to  volume  (200  c.c.)  and  make 
a  quantitative  color  comparison  by  means  of  the  colorimeter. 

Those  who  are  inexperienced  in  colorimetric  work  should  not  fail 
to  observe  the  following  precautions.  Do  not  spill  Nesslerized  or 
other  alkaline  solutions  on  the  mirror  of  the  colorimeter;  a  single 
such  spill  unless  immediately  washed  off  with  an  abundance  of  cold 
water  ruins  the  mirror.  The  most  frequent  cause  of  spilling  is  the 
placing  of  too  much  solution  in  the  colorimeter  cups.  These  cups 
should  be  only  a  little  more  than  half  filled.  Always  rinse  the  colo- 
rimeter cups  and  the  plungers  with  cold  water  after  using,  as  a  red 
sediment  is  otherwise  gradually  deposited  on  both.  Such  sediment, 
when  formed,  can  be  removed  by  means  of  a  dilute  solution  of  po- 
tassium iodide. 

Before  attempting  to  determine  the  color  of  the  unknown  the 
correctness  of  the  instrument,  and  of  the  eye,  must  be  ascertained. 
Rinse  both  colorimeter  cups  with  the  standard  Nesslerized  solution 
and  fill  both  with  the  same  solution  a  little  more  than  half  full.  Put 
them  in  place  and  set  both  colorimeter  plungers  at  a  height  of  ex- 
actly 20  mm.  Adjust  the  focus  of  the  instrument  so  that  the  line 
dividing  the  two  fields  is  clear  and  distinct.  By  the  help  of  the 
mirror  and  by  turning  the  whole  instrument,  adjust  it  to  the  source 
of  light  until  the  two  fields  look  alike.  Do  not  stare  at  the  field  too 
long;  close  the  eye  frequently  so  as  to  avoid  fatigue.  If  the  fields 
can  not  be  made  to  look  alike  the  zero  point  or  the  optics  of  the 
instrument  must  be  wrong  and  suitable  allowance  must  be  made 
for  this  error.  Having  learned  to  see  the  fields  alike,  change  the 
height  of  one  of  the  plungers  and  then  make  a  color  comparison 
of  the  standard  against  itself  readjusting  the  moved  plunger  until 
the  fields  again  look  alike.  The  error  should  not  exceed  0.2  mm. 
Now  set  the  two  plungers  again  at  20  mm.  and  ascertain  once 
more  that  they  look  alike.  Then  empty  one  cylinder,  rinse  it,  and 
also  the  plunger,  with  the  unknown;  half  fill  the  cup  with  the  un- 

101 


known,  and  make  the  final  color  comparison  fairly  rapidly,  before 
the  memory  of  what  the  fields  looked  like  when  they  were  equal 
has  been  blurred. 

The  ammonia  content  is  inversely  proportional  to  the  depth  of 
the  color,  provided  that  one  is  not  more  than  one  and  one-half 
times  as  deep  as  the  other.  Twenty,  the  depth  of  the  standard 
in  mm.,  divided  by  the  reading  of  the  unknown,  in  mm.,  gives 
the  ammonia  nitrogen,  in  mg.,  in  the  volume  of  urine  (or  am- 
monium sulphate  solution)  taken  for  the  analysis. 

Calculate  the  24-hour  quantity  of  the  ammonia  as  NH3  and 
as  ammonia-N,  and  compare  with  the  figures  given  by  the  macro 
aeration  method. 

4.  Clinical  Method  for  the  Determination  of  Ammonia. — The 

special  reagents  required  in  this  determination  are  (a)  saturated 
potassium  oxalate  solution  and  (b)  formalin ;  both  of  which 
must  be  neutral  to  phenolphthalein.  To  each  reagent  add  a  little 
of  the  indicator  and  .1  N  alkali  to  a  faint  pink  coloration. 

To  25  c.c.  of  urine  add  about  5  c.c.  of  the  neutralized  oxalate 
solution  and  2-3  drops  phenolphthalein  solution.  Titrate  the 
acidity  of  the  urine  to  a  faint  but  unmistakable  end  point.  Then 
add  about  5  c.c.  of  the  neutralized  formalin  and  again  titrate  to 
the  same  degree  of  coloration  as  in  the  preceding  titration.  Each 
c.c.  of  the  .1  N  alkali  used  in  this  titration  corresponds  to  I  c.c. 
.1  N  ammonia. 

The  formaldehyd  combines  with  the  ammonia  giving  neutral 
hexamethylenetetramin,  thus  setting  free  acid  equivalent  to  the 
amount  of  ammonia  present. 

tiJL 

5.  Total  Nitrogen.    Colorimetric  Method.  — Special  equipment 
called  for:      (a)   Duboscq  colorimeter,   (b)   standardized  i  c.c. 
"Ostwald  pipets,"  with  extra  long  stems,  (c)  a  solution  of  spe- 
cially purified  ammonium  sulphate,  of  such  strength  that  10  c.c. 
contain  i  mg.  of  nitrogen  (0.4716  g.  salt  per  liter,  (d)  modified 
Nessler-Winkler  reagent  (see  p.  203). 

This  determination  requires  0.7  to  1.5  mg.  of  nitrogen.  The 
total  nitrogen  in  urine  is  on  the  average  about  25  times  as  much 
as  the  "ammonia"  nitrogen.  Dilute  5,  10  or  20  c.c.  of  urine  to 
loo  c.c.,  mix  and  with  an  Ostwald  pipet  transfer  i  c.c.  o'f  the 
diluted  urine  to  a  large  hard  glass  test  tube.  (This  pipet  must 

103 


be  drained  for  15  seconds  against  the  wall  of  test  tube  and  then 
blown  clean.)  With  an  ordinary  pipet  add  I  c.c.  of  the  phos- 
phoric-sulphuric acid-copper  sulphate  mixture  together  with  a 
pebble,  to  prevent  bumping.  Heat  over  a  micro  burner  (no 
hood  necessary)  until  the  water  is  driven  off  and  fumes  become 
abundant  within  the  tube.  This  should  take  place  in  about  two 
minutes.  When  filled  with  fumes  close  the  mouth  of  the  test 
tube  with  a  watch  glass  and  continue  the  boiling  at  such  a  rate 
that  the  tube  remains  filled  with  fumes  yet  almost  none  escape. 
Within  two  minutes  after  the  mouth  of  test  tube  was  closed  the 
contents  should  become  clear,  and  bluish  or  light  green.  Con- 
tinue the  gentle  boiling  for  30  to  60  seconds  longer,  provided, 
however,  that  the  total  boiling  period,  with  test  tube  closed,  must 
not  be  less  than  two  minutes.  Remove  the  flame  and  let  cool  for 
a  little  less  than  two  minutes,  then  add  water.  Rinse  the  hot 
digestion  mixture  (sometimes  turbid  from  silica)  into  a  200  c.c. 
volumetric  flask,  using  for  this  purpose  about  125  c.c.  of  water. 
Transfer  10  c.c.  of  standard  ammonium  sulphate  solution  con- 
taining i  mg.  of  nkrogen  into  another  200  c.c.  volumetric 
flask.  Add  I  c.c.  of  the  concentrated  phosrjhflric-sulphuric 
acid  mixture,  to  balance  the  acid  in  the  unknown,  and  dilute  to  a 

^^L_  f 

volume  of  about  ^p  c.c.  When  both  flasks  are  thus  ready  give 
each  flask  a  whirl  and  add  30  c.c.  of  Nessler's^Psagent.  Slfake  a 
little  more  and  dilute  both  flasks  to  the  200  c.c.  mark. 

If  the  unknown  Nesslerized  digestion  mixture  is  turbid,  centri- 
fuge a  portion,  giving  a  crystal  clear  fluid  above  a  white  sediment 
(siliqa^).  If  the  sediment  is  colored  the  Nesslerization  was  not 
successful  and  the  determination  must  be  discarded.  Determine 
the  color  value  of  the  centrifuged  solution  as  described  under  the 
permutit  method  for  determining  ammonia  and  calculate  the  total 
nitrogen  in  the  three-hour  or  twenty-four  hour  quantity  of  urine. 

Determine  the  nitrogen  in  5  c.c.  of  the  undiluted  urine  by  the 
macro  Kjeldahl  process  and  compare  with  the  value  given  by 
the  colorimetric  process.  The  two  methods  should  give  sub- 
stantially identical  values. 

6.  Reactions  of  Urea. — Put  a  crystal  of  urea  on  a  glass  slide 
or  a  watch-glass  and  cover  it  with  a  drop  of  water.  With  a  glass 
rod,  put  a  drop  of  nitric  acid  next  to  this.  Let  the  two  drops 
run  together,  and  notice  the  precipitation  of  urea  nitrate  at  the 
junction,  Examine  under  the  microscope,  and  sketch  the  crystals. 

105 


Put  a  few  crystals  of  urea  into  a  dry  test  tube,  and  heat  till 
they  melt.  With  moist  litmus  paper  test  the  reaction  of  the  fumes 
given  off.  Explain. 

Cool  the  test  tube.  To  the  residue,  consisting  of  biuret  and 
cyanuric  acid,  add  a  little  water,  filter,  and  with  the  filtrate  make 
the  biuret  test. 

Dissolve  a  few  crystals  of  urea  in  5  c.c.  water  in  a  test  tube. 
Test  its  reaction  with  litmus  paper.  Heat  the  solution  to  boiling 
and  test  the  steam  with  moist  litmus  paper.  Cool  the  liquid  and 
test  with  litmus  paper.  Explain. 

To  a  solution  of  urea  in  a  test  tube  add  an  equal  volume  of 
sodium  hypobromite  solution.  Make  this  by  mixing  and  cooling 
equal  volumes  of  bromin  solution  and  40  per  cent,  sodic  hydrate 
solution. 

The  reaction  with  sodium  hypobromite  has  been  used  for  the 
quantitative  determination  of  urea,  but  as  ordinarily  used  for  this 
purpose  the  method  has  very  little  value. 

7.  Colorimetric  Method  for  Determination  of  Urea  (/.  Biol. 
Chem.  26,  501,  1916;  38,  in,  1919). — Merck's  blood  charcoal  was 
a  necessary  reagent  in  the  determination  of  urea  in  urine  by  the 
direct  Nesslerization  process  of  Folin  and  Denis.  By  using 
urease  preparations  sufficiently  free  from  nitrogenous  materials 
the  urea  nitrogen  can  be  Nesslerized  without  any  charcoal  treat- 
ment. 

Urease  Preparation. — Wash  about  3  g.  of  permutit  in  a  flask 
once  with  2  per  cent,  acetic  acid,  then  twice  with  water ;  add  5  g. 
of  fine  Jack  bean  meal  and  100  c.c.  of  15  per  cent,  alcohol  (16  c.c. 
of  ordinary  alcohol  plus  84  c.c.  of  water).  Shake  gently  but 
continuously  for  10  to  15  minutes,  pour  on  a  large  filter  and  cover 
with  a  watch  glass.  The  filtrate  contains  practically  the  whole 
of  the  urease  and  extremely  little  of  other  materials.  The  urease 
solution  will  keep  for  about  a  week  at  room  temperatures  and 
for  4  to  6  weeks  in  an  ice  box. 

Buffer  Mixtures  for  Urease  Decompositions. — Mixtures  of 
mono-  and  disodium  phosphates  in  the  proportion  I  molecule  of 
the  former  to  2  of  the  latter,  and  in  molar  concentrations,  are 
usually  employed  to  preserve  a  substantially  neutral  reaction 
during  the  decomposition  of  urea  by  means  of  urease.  Dissolve 
69  g.  of  monosodium  phosphate  and  179  g.  of  crystallized  di- 

107 


sodium  phosphate  in  800  c.c.  of  warm  distilled  water.  Cool  and 
dilute  to  a  volume  of  i  liter. 

It  would  seem  to  be  rather  doubtful  whether  the  maintenance 
of  neutrality  is  adequate  to  fully  explain  the  accelerating  action 
of  phosphates  on  the  urea  decomposition,  because  pyro-  and 
metaphosphates  seem  to  be  more  effective  than  orthophosphates. 
An  excellent  buffer  mixture  is  obtained  by  dissolving  14  g.  of 
sodium  pyrophosphate  (Na4P2O7,  ioH2O)  in  enough  half  nor- 
mal phosphoric  acid  to  make  a  volume  of  100  c.c.  The  half  nor- 
mal phosphoric  acid  is  made  by  diluting  20  c.c.  of  85  per  cent, 
phosphoric  acid  to  I  liter  and  titrating  5  c.c.  with  tenth  normal 
alkali  and  phenolphthalein  as  indicator  to  a  faint  pink  color.  On 
the  basis  of  this  titration  dilute  the  acid  to  a  substantially  correct 
half  normal  solution.  Metaphosphoric  acid  is  fully  as  good  as 
phosphoric  acid,  but  it  is  much  more  difficult  to  prepare  a  solution 
of  the  requisite  degree  of  neutrality  with  metaphosphoric  acid 
because  of  its  variable  water  and  free  phosphoric  acid  content. 
The  pyrophosphate-phosphoric  acid  mixture,  the  preparation  of 
which  is  described  above,  gives  a  faint  color  with  rosalic  acid. 
5  c.c.  when  titrated  with  tenth  normal  alkali  and  phenolphthalein 
should  give  a  faint  but  distinct  color  with  about  18  c.c.  of  the 
alkali. 

Transfer  with  an  Ostwald  pipet  I  c.c.  of  diluted  urea  solution 
or  urine  (dilution  sometimes  5  or  20,  but  usually  10  c.c.  diluted 
to  100  c.c.)  to  a  clean  test  tube;  add  i  or  at  the  most  2  drops  of 
buffer  mixture  and  i  c.c.  of  urease  solution.  Digest  in  a  beaker 
of  warm  tap  water  (40°  to  55°  C.)  for  5  minutes  or  at  room 
temperatures  for  15  minutes.  At  the  end  of  the  digestion  period 
rinse  the  contents  of  the  test  tube  into  a  200  c.c.  volumetric  flask 
and  dilute  to  a  volume  of  about  150  c.c. 

Transfer  i  mg.  of  N  in  the  form  of  ammonium  sulphate  to 
another  200  c.c.  volumetric  flask;  to  this  standard  add  I  c.c.  of 
urease  solution  and  dilute  to  about  150  c.c.  Then  add  with  shak- 
ing (with  a  cylinder)  20  c.c.  of  Nessler  solution  to  each.  Dilute 
to  volume  and  make  the  color  comparison,  never  omitting  to  first 
read  the  standard  against  itself. 

The  height  of  the  standard  (usually  20  m.m.)  divided  by  the 
height  of  the  unknown,  gives  the  nitrogen,  in  mg.,  present  in  the 
fraction  of  a  c.c.  of  undiluted  urine  present  in  the  i  c.c.  of 
diluted  urine  taken  for  the  analysis. 

Unless  the  colorimetric  reading  is  between  14  mm.  and  30  mm. 

109 


the  determination  should  be  repeated  with  i  c.c.  of  urine  so 
diluted  as  to  give  readings  coming  within  those  limits.  Calculate 
the  total  urea-N  and  subtract  the  preformed  ammonia-N. 

A  few  explanatory  remarks  may  be  added.  Many  kinds  of  bio- 
logical nitrogenous  materials,  particularly  ammo  acids,  peptones  and 
albumins,  prevent  the  development  of  the  color  reaction  given  by 
ammonia  and  Nessler's  reagent.  This  was  first  discovered  in  attempts 
to  determine  by  direct  Nesskrization  the  ammonia  formed  in  pan- 
creatic digestion  mixtures.  If  very  little  such  nitrogenous  material 
is  present  the  result  obtained  is  deceptive  for  then -the  color  is 
merely  diminished  and  the  error  will  not  be  detected.  The  careful 
observer  will  find,  however,  that  in  such  cases  the  color  obtained 
.tends  to  be  visibly  more  greenish  and  less  distinctly  red  than  the 
standard.  Because  of  the  serious  interference  caused  by  albuminous 
materials  it  may  be  thought  that  the  procedure  described  above  is  not 
applicable  to  albuminous  urines,  but  a  series  of  determinations  have 
shown  that  even  urines  very  rich  in  albumin  have  in  fact  so  little  in 
comparison  with  the  amount  of  urea  present  that  correct  results  are 
invariably  obtained  by  direct  Nesslerization. 

Because  of  the  extremely  low  nitrogen  content  of  our  urease  prep- 
aration it  is  not  really  essential  that  the  urease  should  also  be  added 
to  the  standard  ammonia  solution,  but  we  have  thought  it  best  *o 
recommend  that  it  be  added  simply  as  a  precaution  against  the  pos- 
sible occurrence  of  less  good  urease  preparations.  In  recommending 
the  addition  of  the  urease  to  the  standard  as  well  as  to  the  urine 
we  have  also  had  in  mind  the  probability  that  some  will  omit  the 
use  of  the  permutit  when  making  the  alcoholic  urease  extracts  and 
will  then  have  variable  small  traces  of  ammonia  in  their  extracts. 

The  reason  why  the  urease  decomposition  had  best  be  made  in 
test  tubes  rather  than  in  volumetric  flasks  is  to  avoid  failure  due  to 
the  use  of  flasks  which  have  been  used  for  Nesslerization  purposes. 
Such  flasks  may  look  perfectly  clean,  but,  unless  they  have  been 
rinsed  with  nitric  acid,  they  will  contain  enough  mercury  compounds 
to  destroy  entirely  the  urease  and  scarcely  a  trace  of  ammonia  is 
obtained. 

Compare  the  urea-N  value  of  the  urea  solution  with  that  ob- 
tained by  the  Macro  Kjeldahl  process. 

8. '  Uric  Acid  Preparation  of  Uric  Acid  from  Urine.  —  To  500 

c.c.  of  urine  add  25  g.  of  ammonium  sulphate,  stir  until  the  sul- 
phate is  dissolved,  and  add  about  10  c.c.  of  ammonia.  Let  stand 
over  night,  filter,  and  wash  two  or  three  times  with  water  con- 
taining a  little  ammonia.  Transfer  the  precipitate  to  a  small 

111 


beaker,  add  a  few  drops  of  hydrochloric  acid,  and  let  stand  till 
the  following  day.  Examine  the  crystals  under  the  microscope. 

9.  Murexid  Test  for  Uric  Acid. — Place  a  few  uric  acid  crystals 
on  a  porcelain  crucible  cover.     Add  three  drops  of  strong  nitric 
acid.     Heat  cautiously,  blowing  on  the  liquid  to  complete  dry- 
ness.    A  red  color  should  appear.    Let  cool  and  add  a  few  drops 
of  dilute  ammonia.    Repeat  with  cafrein.    Explain. 

10.  Phosphotungstic  Acid  Test  for  Uric  Acid. — Dissolve  a  few 
crystals  of  uric  acid  in  2  c.c.  very  dilute  sodic  hydrate  solution. 
Add  i  c.c.  of  "qualitative  uric  acid  reagent"  (see  p.  207).    Then 
add    10  c.c.   of   saturated   sodium   carbonate   solution.     A   pro- 
nounced blue  coloration  should  be  obtained.    Repeat  the  reaction 
with  5  c.c.  of  urine. 

11.  Colorimetric  Method  for  the  Determination  of  Uric  Acid. 

(Journal  of  Biol.  Chem.  38,  1919). —  Transfer  1-3  c.c.  of  urine, 
according  to  concentration,  to  a  centrifuge  tube  and  add  water 
to  a  volume  of  about  6  c.c.  Add  5  c.c.  of  a  silver  lactate  solution 
(silver  lactate  5  per  cent.,  lactic  acid  5  per  cent.),  and  stir  with 
a  fine  glass  rod.  Rinse  off  the  rod  with  a  few  drops  of  water. 
Centrifuge  the  counterbalanced  tube  for  2-3  minutes.  Add  a 
drop  of  silver  lactate  solution  so  as  to  be  sure  that  an  excess  is 
present;  if  a  precipitate  (of  AgCl)  is  formed,  add  2  c.c.  more  of 
the  silver  solution  and  centrifuge  again ;  if  no  precipitate  forms, 
pour  off  the  liquid  as  completely  as  possible. 

To  the  precipitate  in  the  centrifuge  tube  add,  from  a  buret, 
4  c.c.  of  a  5  per  cent,  sodium  cyanid  solution  (poisonous — 3  c.c. 
may  be  fatal  dose).  Stir  the  mixture  until  a  perfectly  clear  solu- 
tion is  formed.  Rinse  the  stirring  rod,  collecting  the  rinsings  in 
a  100  c.c.  graduated  flask ;  pour  the  contents  of  the  tube  into  the 
same  flask  and  rinse  the  tube  3  times  with  about  5  c.c.  of  water. 
Add  5  c.c.  of  a  10  per  cent,  sodium  sulphite  solution  (to  balance 
the  sulphite  in  the  standard  uric  acid  solution)  and  dilute  to  a 
volume  of  about  40  c.c.  In  another  100  c.c.  flask  place  5  c.c.  of 
a  standard  uric  acid-sulphite  solution  (see  below)  containing  0.5 
mg.  of  uric  acid ;  add  4  c.c.  of  cyanid  solution  and  about  35  c.c. 
of  water.  Then  add  20  c.c.  of  20  per  cent,  sodium  carbonate 
solution  to  each  flask  and  finally  add  with  shaking  2  c.c.  of  the 
uric  acid  reagent  described  on  p.  207.  Let  stand  3-5  minutes, 
fill  to  the  mark  and  mix. 

113 


Set  the  standard  uric  acid  solution  at  20  m.m.  in  both  colo- 
rimeter cups  and  adjust  the  colorimeter  until  the  two  fields  are 
exactly  alike.  Then  determine  the  color  of  the  unknown.  Since 
the  standard  is  only  0.5  mg.,  10  divided  by  the  reading  of  the 
unknown  (in  mm.)  gives  the  amount  of  uric  acid  (in  mg.)  in 
the  volume  of  urine  taken. 

Caution, — Be  careful  to  pour  the  discarded  blue  uric  acid 
cyanid  mixtures  directly  into  the  drain  pipes  of  sinks.  If  sinks 
contain  acids  a  gaseous  mixture  of  C02  and  HNC  will  be  set 
free. 

The  uric  acid  reagent  gives  an  intense  blue  color  with  uric  acid 
in  the  presence  of  an  alkali.  The  same  blue  color  is  obtained 
with  some  other  substances,  notably  with  some  phenol  derivatives 
present  in  urine.  It  is  therefore  necessary  to  separate  the  uric 
acid  from  these  products  before  applying  the  color  reaction.  Acid- 
ified silver  lactate  is  used  for  the  precipitation  of  the  uric  acid. 
In  the  presence  of  sodium  chlorid  it  carries  down  every  trace 
of  uric  acid,  provided  that  an  excess  of  the  silver  salt  is  added. 
The  sodium  cyanid  dissolves  the  silver  precipitate  and  sets  free 
the  uric  acid;  it  also  greatly  reduces  the  tendency  of  the  color 
to  fade  on  standing.  The  use  of  the  cyanid  first  introduced  by 
S.  R.  Benedict  has  greatly  simplified  the  uric  acid  determination. 

Preparation  of  Standard  Uric  Acid  Solution. — In  a  500  c.c. 
flask  dissolve  exactly  I  g.  of  uric  acid  in  150  c.c.  of  water  by 
the  help  of  0.5  g.  lithium  carbonate.  Dilute  to  500  c.c.  and  mix. 
Transfer  50  c.c.  to  a  liter  flask;  add  500  c.c.  of  20  per  cent, 
sodium  sulphite  solution ;  dilute  to  volume  and  mix.  Transfer  to 
small  bottles  (cap.  200  c.c.)  and  stopper  tightly.  This  standard 
uric  acid  solution  keeps  almost  indefinitely  in  unopened  bottles, 
because  the  sulphite  prevents  the  spontaneous  oxidation  of  the 
uric  acid.  In  used  bottles  the  standard  usually  remains  good  for 
2-3  months. 

12.  Creatinin.— Transfer  to  a  small  bottle  aboft  3  g.  dry  picric 
acid.  Add  about  100  c.c.  of  urine,  insert  a  cork;  and  shake  con- 
tinuously for  ten  minutes.  Filter  a  portion  of  the  mixture  and 
transfer  5  c.c.  of  the  filtrate  to  a  bottle  or  flask  (capacity  not  less 
than  250  c.c.). 

Measure  5  c.c.  of  the  original  urine  into  another  similar  flask 
,or  bottle.  Add  to  each,  first  20  c.c.  saturated  picric  acid  solvt- 

115 


tion,  and  then  5  c.c.  10  per  cent,  sodic  hydrate.  Let  stand  5-10 
minutes  and  add  200  c.c.  tap  water  to  each. 

Remove  by  decantation  the  liquid  from  the  bottle  containing 
urine  and  picric  acid,  and  to  the  sediment  add  about  25  c.c.  water 
and  15  c.c.  sodic  hydrate  (10  per  cent.).  Shake,  let  stand  for  a 
few  minutes,  and  fill  the  bottle  with  tap  water. 

The  substance  in  urine  which  is  precipitated  by  picric  acid  and 
which  gives  a  deep  red  color  with  alkaline  picrate  solutions, 
is  creatinin.  No  other  known  substance  occurring  in  normal 
urine  gives  this  color  reaction.  Therefore,  on  the  basis  of  this 
reaction,  it  is  easy  to  determine  the  creatinin  quantitatively. 

13.  Quantitative  Determination  of  Creatinin.— A  suitable  and 
convenient  "ereatinin  reagent"  is  prepared  by  adding  75  c.c.  of 
10  per  cent,  sodic  hydrate  to  a  liter  of  saturated  picric  acid  solu- 
tion. If  the  picric  acid  is  pure  and  the  alkaline  solution  is  kept 
away  from  the  light  and  from  dust  it  keeps  well  for  several  days. 
It  is  usually  more  safe,  however,  to  prepare  only  so  much  of  the 
solution  as  is  used  up  the  same  day.  For  a  single  determination 
it  is  not  worth  while  to  prepare  the  reagent;  employ  instead  the 
picric  acid  solution  and  the  alkali,  using  20  c.c.  of  the  former  and 
15  c.c.  of  the  latter. 

By  means  of  an  Ostwald  pipet  transfer  i  c.c.  or  2  c.c.  urine  to 
a  100  volumetric  flask.  To  another  similar  flask  transfer  I  c.c. 
of  a  standard  creatinin  solution  (1.61  g.  of  creatinin  zinc  chlorid 
dissolved  in  one  liter  of  tenth  normal  hydrochloric  acid),  I  c.c. 
of  which  contains  I  mg.  of  creatinin.  To  each  flask  add  20  c.c. 
of  picric  acid  solution,  then  add  from  a  buret  1.5  c.c.  of  10  per 
cent,  sodic  hydrate  to  each,  and  let  stand  for  ten  minutes.  If  the 
alkaline  picrate  solution  is  used,  add,  with  a  cylinder,  20  c.c.  to 
each  flask.  At  the  end  of  ten  minutes  dilute  to  the  mark  with 
water  and  mix. 

Read  the  standard  against  itself  in  the  colorimeter  at  20  mm. 
until  the  correct  value  (20  mm.)  can  be  obtained.  The  error  in 
reading  should  not  exceed  .2  mm. .  Rinse  the  right-hand  cup  and 
prism  with  the  unknown,  and  determine  its  color  in  terms  of  the 
standard  set  at  20  mm.  Twenty  divided  by  the  reading  gives  the 
creatinin  in  milligrams  in  the  quantity  of  urine  taken  (1-5  c.c.). 

Calculate  the  total  creatinin. 

-  14.  Creatin. — Unless  considerable  meat  or  fish  has  been  eaten 
the  urine  of  normal  adults  contains  only  traces  of  creatin. 

117 


Urines  of  children  and  of  sick  persons,  particularly  fever  pa- 
tients, appear,  on  the  other  hand,  to  contain  relatively  consider- 
able quantities  of  creatiri  (.2  g.  to  i  g.  or  more  per  day  in  fever 
patients). 

Creatin  is  determined  in  such  urines  by  first  converting  it  into 
creatinin.  This  is  done  as  follows: 

Measure  the  urine  (usually  i  c.c.)  into  a  flask  (capacity  300 
c.c.)  and  add  20  c.c.  saturated  picric  acid  solution  (not  the 
creatinin  reagent).  Weigh  flask  and  contents  and  add  about  150 
c.c.  water.  Boil  gently  for  45  minutes,  then  more  rapidly  until 
the  original  volume  (determined  by  weighing)  is  obtained  (a  vari- 
ation of  3  or  4  g.  makes  no  appreciable  difference).  Cool.  Add 
1.5  c.c.  10  per  cent,  sodic  hydrate,  let  stand  10  minutes,  and  com- 
pare, as  in  the  case  of  preformed  creatinin,  with  the  color  ob- 
tained from  i  mg.  creatinin. 

Twenty  divided  by  the  reading  gives  the  sum  of  the  creatin  and 
creatinin  present. 

Calculate  the  total  quantity  and  subtract  the  preformed  cre- 
atinin. 

If  an  autoclave  is  available,  the  conversion  into  creatin  can  be 
made  more  rapidly. 

Measure  the  urine  (i  c.c.)  into  a  100  c.c.  volumetric  flask, 
and  add  20  c.c.  saturated  picric  acid  solution.  Cover  the  mouth 
of  the  flask  with  tinfoil,  and  heat  in  the  autoclave  at  H5°-I2O° 
for  20  minutes.  Cool,  add  1.5  c.c.  sodic  hydrate,  and  finish  the 
determination  in  the  usual  manner. 

*  15.     Hippuric  Acid'.— Take  with  the  evening  meal  2  g.  of  so- 
dium benzoate,  and  collect  the  urine  until  next  morning. 

Evaporate  to  small  volume  and  transfer  with  a  little  wash 
water  to  a  small  flask.  Acidify  strongly  with  sulphuric  acid  and 
put  away  for  twenty-four  hours.  Filter  and  dry  the  precipitate, 
consisting  of  hippuric  acid,  uric  acid,  and  other  substances.  Ex- 
tract the  hippuric  acid  with  acetic  ether.  Set  aside  for  sponta- 
neous evaporation.  Examine  microscopically.  Heat  the  dry  sub- 
stance in  a  dry  tube,  and  note  the  odor  of  bitter  almonds  (benzal- 
dehyd). 

~ 

16.  Determination  of  Inorganic  Sulphates. —  (/.  Biol.  Chem., 
i,  131). — Transfer  to  a  250  c.c.  beaker  25  c.c.  of  the  ammonium 
sulphate  solution  in  which  the  ammonia  was  determined  (p.  23). 

119 


Dilute  with  water  to  about  100  c.c. ;  add  10  c.c.  of  20  per  cent, 
sodium  chlorid  solution  and  5  c.c.  of  concentrated  hydrochloric 
acid. 

The  addition  of  sodium  chlorid  is  necessary  only  when  the  sulphate 
determination  is  to  be  made  in  ammonium  or  potassium  sulphate 
solutions.  Such  solutions  give  too  low  sulphate  values  unless  sodium 
chlorid  is  added.  The  reason  is  rather  obscure,  but  in  general  it  may 
be  stated  that  the  sulphate  piecipitate  obtained  is  practically  never 
pure  BaSO4,  but  by  adding  sodium  chlorid  the  precipitate  obtained 
has  been  found,  empirically,  to  give  the  weight  corresponding  to 
BaSO4.  Urine  contains  so  much  more  of  sodium  than  of  ammonium 
and  potassium  salts  that  in  urine  analysis  the  addition  of  sodium 
chlorid  is  not  required. 

Fill  a  buret  with  5  per  cent,  barium  chlorid  solution  and  place 
the  beaker,  containing  the  sulphate  solution,  under  it  so  that  the 
buret  will  deliver  on  the  side  (the  spout)  of  the  beaker.  Add 
thus,  drop  by  drop,  10  to  15  c.c.  of  barium  chlorid.  The  contents 
in  the  beaker  must  not  be  stirred  or  agitated  to  any  degree  while 
the  barium  is  added.  If  BaSO4  is  formed  too  rapidly  in  the 
cold  much  "occlusion"  takes  place,  and,  in  addition,  the  precipi- 
tate is  apt  to  be  so  fine  that  it  will  pass  through  when  filtering. 
After  the  beaker  has  stood  for  10  to  30  minutes  mixing  the 
contents  of  the  beaker  will  do  no  harm  and  must  be  done  to 
complete  the  precipitation.  Let  stand  for  i  to  24  hours,  accord- 
ing to  convenience,  before  filtering.  (The  ethereal  sulphates  are 
not  hydrolyzed  in  the  cold.) 

Prepare  an  asbestos  mat  as  follows:  Fill  the  Gooch  crucible 
with  the  freshly  shaken  asbestos  suspension  once,  or  at  the  most 
twice.  Pack  the  asbestos  down  by  vigorous  suction  (water 
pump).  The  mat  should  be  about  i  mm.  thick.  Then  cover  the 
mat  with  a  perforated  porcelain  plate  or  with  a  layer  of  sea  sand, 
5  to  7  mm.  thick.  The  sand  should  previously  be  kept  for  at 
least  24  hours  in  contact  with  5  per  cent,  hydrochloric  acid. 
Wash  the  asbestos  and  covering  with  water  and  moderate  suc- 
tion until  the  filtrate  is  free  from  asbestos  particles  and  water 
clear.  Make  as  dry  as  possible  by  suction.  Heat  very  gently  at 
first  (not  over  100°  C.)  to  drive  off  the  steam  without  mechani- 
cal disturbance  of  the  mat;  then  ignite ;  cool  for  15  to  20  minutes, 
and  weigh.  The  same  mat  can  be  used  for  several  successive 
.sulphate  determinations  provided  that  adequate  care  is  taken  not 

121 


to  disturb  the  mat,  either  by  too  rapid  use  of  water  when  filter- 
ing or  by  steam  during  the  heating. 

Transfer  the  barium  sulphate  to  the  Gooch  crucible  and  wash 
6  to  10  times  with  distilled  water.  Heat  to  dryness,  ignite,  cool 
and  weigh.  From  the  weight  of  the  BaSO4  obtained,  calculate 
the  ammonium  sulphate  and  compare  with  the  weight  known  to 
be  present.  Repeat  with  25  c.c.  of  urine  to  which  has  been  added 
about  75  c.c.  of  water  and  5  c.c.  of  hydrochloric  acid.  (No 
sodium  chlorid  need  be  added  when  working  with  urine.)  Cal- 
culate the  results  as  S  and  as  H2SO4. 

P"  17.  Determination  of  Total  Sulphates. — In  this  determination 
the  ethereal  sulphates  must  be  hydrolyzed  by  hydrochloric  acid 
and  heat  before  barium  chlorid  is  added.  Transfer  25  c.c.  of 
urine  to  a  beaker,  add  about  25  c.c.  of  water  and  5  c.c.  of  con- 
centrated hydrochloric  acid.  Cover  with  a  watch  glass  and  boil 
gently  for  20  to  30  minutes.  Then  dilute  to  about  100  c.c.,  heat 
to  boiling  and  with  a  pipet  add  10  c.c.  of  5  per  cent,  barium 
chlorid  solution.  Let  stand  for  an  hour,  or  as  much  longer  as 
may  be  convenient.  Filter,  wash,  ignite,  cool  and  weigh,  as  in 
the  case  of  the  inorganic  sulphate  determination. 

Calculate  as  S  and  as  H2SO4  and,  by  subtraction  of  the  corre- 
sponding values  obtained  as  inorganic  sulphates,  calculate  the  S 
and  H2SO4  present  in  the  form  of  ethereal  sulphates. 

t£  18.  Determination  of  Total  Sulphur. —  (Benedict,  /.  Biol. 
Chem.,  6,  363 ;  Denis,  /.  Biol.  Chem.,  8,  401.) — Transfer  25  c.c.  of 
urine  to  a  porcelain  dish  (diam.  10-12  cm.)  ;  add  5  c.c.  of  a  solu- 
tion containing  25  per  cent,  of  copper  nitrate  and  25  per  cent,  of 
sodium  chlorid,  and  10  per  cent,  of  ammonium  nitrate.  Evap- 
orate to  dryness  on  the  water  bath.  Then  heat  over  a  flame, 
preferably  over  a  Fletcher  burner;  the  burners  attached  to  the 
copper  condensers  used  in  Kjeldahl  determinations  are  also  good. 
The  heat  should  be  very  moderate  at  first  and  should  then  be 
gradually  increased  until  the  dish  becomes  almost  red  hot,  con- 
tinue the  vigorous  heating  for  10  minutes  so  as  to  decompose 
and  drive  off  all  of  the  nitric  acid  fumes.  The  organic  matter, 
including  the  sulphur  compounds,  are  thus  oxidized,  but  the  sul- 
phates formed  do  not  escape;  they  are  held  back  as  copper 
sulphate.  The  sodium  chlorid  present  in  the  oxidizing  nitrate 
mixture  serves  to  prevent  mechanical  losses  due  to  the  explosive 

123 


violence  with  which  the  oxidations  are  apt  to  occur  without  the 
presence  of  the  sodium  chlorid.  Allow  the  dish  to  cool.  Add 
20  c.c.  of  10  per  cent,  hydrochloric  acid  and  warm  quickly. 
Filter  the  dissolved  contents  into  a  beaker,  using  for  this  purpose 
75  to  100  c.c.  of  hot  water.  Heat  to  boiling  and  add  slowly  15 
c.c.  of  5  per  cent,  barium  chlorid  solution.  Let  stand  an  hour 
or  longer,  filter  on  the  asbestos  mat,  wash,  ignite,  cool  and  weigh. 
Calculate  as  S  and  as  H2SO4  and  by  subtraction  of  the  S  and 
H2SO4  found  as  total  sulphates,  calculate  the  values  correspond- 
ing to  the  "neutral"  or  "unoxidized"  sulphur. 

19.     Phosphates.—  Add  a  few  drops  neutral  calcium  chlorid 
solution  : 

(a)  to  5  c.c.  i  per  cent,  monopotassium  phosphate  solution, 

(b)  to  5  c.c.  i  per  cent,  disodium  phosphate  solution, 

(c)  to  a  mixture  of  the  two  phosphate  solutions, 

(d)  to  5  c.c.  turbid  urine, 

(e)  to  5  c.c.  clear  urine, 

(f)  to  5  c.c.  of  urine   after  filtering  off  the  precipitate  ob- 
tained by  the  addition  of  a  little  ammonia.    Explain  the  results. 


Determination  of  Phosphates.  —  STANDARD  PHOSPHATE  SO- 
LUTION. —  Dissolve  4.39  g.  pure  monopotassium  phosphate  in 
water  and  dilute  to  500  c.c.  Each  c.c.  contains  2  mg.  phosphorus. 
Label  and  preserve. 

STANDARD  URANIUM  SOLUTION.  —  Dissolve  18  g.  uranium  ace- 
tate and  50  c.c.  50  per  cent,  acetic  acid  in  water.  Dilute  to  500 
c.c.  If  turbid,  allow  to  settle  (for  a  day  or  two),  and  remove 
the  clear  supernatant  solution  by  means  of  a  siphon. 

Transfer  25  c.c.  of  the  phosphate  solution  to  a  flask,  add  5  c.c. 
special  sodic  acetate  solution  (containing  10  per  cent,  acetate  and 
3  per  cent,  acetic  acid),  heat  to  boiling,  and  add  15-20  c.c.  of  the 
clear  uranium  solution  from  a  buret.  Heat  again  to  boiling,  and 
now  add  the  uranium  slowly  until  2  drops  of  the  phosphate 
uranium  mixture  when  added  (by  means  of  a  glass  tube  drawn 
out  to  a  point  like  a  pipet)  to  a  minute  pinch  of  powdered  potas- 
sium ferrocyanid  (on  a  white  plate)  gives  a  faint  yet  unmis- 
takable brownish  coloration. 

Repeat  until  the  exact  titrating  value  of  the  uranium  solution 
has  been  ascertained. 

Calculate  the  value  of  the  uranium  solution  in  terms  of  phos- 
phorus (P)  and  also  as  H3PO4.  Label  and  preserve, 

125 


DETERMINATION  OF  PHOSPHATES  IN  URINE. — Measure  50  c.c. 
of  urine  into  a  flask,  add  sodic  acetate,  heat  to  boiling,  and  titrate 
with  the  uranium  solution,  exactly  as  in  the  case  of  the  standard 
phosphate  solution.  After  having  found  the  approximate  phos- 
phate content  by  means  of  the  preliminary  titration,  repeat,  add- 
ing nearly  all  the  required  uranium  solution  at  once  to  the  hot 
urine,  and  finish  by  adding  only  a  few  drops  at  a  time. 

Calculate  in  terms  of  phosphorus  and  of  phosphoric  acid  the 
phosphate  content  of  the  24  hour  urine  under  examination. 

21.  Acidity  of  Urine.— (See  Am.  Journ.  of  Physiology,  9, 
265,  1903,  and  13,  102,  1905.) 

Nearly  all  of  the  titratable  acidity  of  urine  is  due  to  acid 
phosphate.  The  end  point  of  this  titration  is  not  very  sharp, 
partly  on  account  of  the  color  of  the  urine  (compare  p  91) 
and  partly  because  of  the  presence  of  ammonium  salts.  The 
titration  is  further  complicated  by  the  presence  of  calcium,  for 
when  alkali  is  added  to  a  mixture  of  phosphate  and  calcium 
some  tribasic  phosphate  is  precipitated.  In  the  presence  of 
sodium  or  potassium  oxalate  the  premature  formation  of  tri- 
basic salt  is  prevented.  But,  on  account  of  the  ammonium  salts, 
there  is  still  danger  of  over-titrating  and  the  first  distinguishable 
coloration  should  be  taken  as  the  end  point.  With  phenolphtha- 
lein  as  indicator  titrate  the  acidity  of  the  monopotassium  phos- 
phate (20  c.c.)  used  for  standardizing  the  uranium  solution. 

Repeat  after  having  added  5  c.c.  of  neutral  calcium  chlorid 
solution  (2  per  cent). 

Repeat  after  having  added  5  c.c.  of  the  calcium  chlorid  solu- 
tion and  5  c.c.  of  saturated  neutralized  potassium  oxalate  solu- 
tion. 

Note. — For  accurate  work  15  g.  of  solid  neutral  potassium  oxalate 
should  be  used  instead  of  the  oxalate  solution  prescribed,  but  it  is 
rather  difficult  to  obtain  strictly  neutral  oxalate. 

Transfer  20  c.c.  of  undiluted  urine  to  a  small  flask  or  beaker, 
add  5  c.c.  of  the  oxalate  solution.  With  another  sample  of  the 
same  urine  in  another  flask  or  beaker  as  a  guide,  and  with 
phenolphthalein  as  indicator,  titrate  until  the  oxalated  urine 
becomes  a  shade  darker  than  the  other. 

Calculate  the  acidity  for  the  whole  urine  in  terms  of  tenth 
normal  acid. 

127 


Calculate  the  phosphate  of  the  same  urine  in  terms  of  tenth 
normal  phosphoric  acid,  but  considering  the  H3PO4  as  a  mono- 
basic acid.  Compare  the  two  values  and  calculate  what  per  cent, 
of  the  phosphate  is  present  as  acid  phosphate.  The  remainder, 
if  any,  is  dibasic  phosphate. 

Compare  also  the  acidity  and  the  acid  phosphate  with  the 
ammonia,  expressed  in  c.c.  of  tenth  normal  solution.  The  am- 
monia usually  varies  more  or  less  directly  with  the  acid  phos- 
phate (and  the  acidity). 

22,  Determination  of  Chlorids.— STANDARD  SILVER  NITRATE 
SOLUTION. — This  is  prepared  by  dissolving  23.94  g.  silver  nitrate 
per  liter  of  solution  (or  5.99  g.  in  250  c.c.). 

STANDARD  AMMONIUM  SULPHOCYANATE  SOLUTION. — Dis- 
solve 6  g.  of  the  salt  in  800-900  c.c.  water.  Transfer  10  c.c.  of 
the  silver  solution  to  a  beaker  or  flask ;  add  50  c.c.  water,  5  c.c. 
concentrated  nitric  acid,  and  2  c.c.  of  saturated  ferric  ammo- 
nium sulphate  solution.  By  means  of  a  buret  titrate  the  acidified 
silver  solution  with  the  sulphocyanate  solution.  On  the  basis  of 
the  result,  dilute  a  part  of  the  sulphocyanate  solution  so  as  to 
give  500  c.c.  (or  a  liter)  of  a  solution,  20  c.c.  of  which  is  exactly 
equivalent  to  10  c.c.  of  the  silver  solution. 

Each  c.c.  of  the  silver  solution  corresponds  to  5  mg.  chlorin 
(or  to  8.23  mg.  sodium  chlorid). 

The  chlorin  determination  in  urine  is  carried  out  as  follows : 

Pipet  10  c.c.  of  urine  into  a  100  c.c.  volumetric  flask,  add  50 
c.c.  distilled  water,  5  c.c.  saturated  ferric  alum  solution,  and  5  c.c. 
concentrated  nitric  acid.  Add  20  c.c.  standard  silver  nitrate  solu- 
tion, fill  up  to  the  mark  with  distilled  water,  and  shake.  Filter 
through  a  dry  filter  into  a  dry  beaker  or  flask.  With  a  clean,  dry 
pipet  transfer  50  c.c.  of  the  filtrate  to  another  beaker,  flask,  or 
evaporating  dish,  and  titrate  in  the  same  manner  as  when  stand- 
ardizing the  silver  solution. 

Since  the  sulphocyanate  solution  is  half  as  strong  as  the  silver 
solution,  and  since  only  one-half  of  the  surplus  silver  was  taken 
for  titration,  20  minus  the  sulphocyanate  titration  figure  repre- 
sents the  silver  nitrate  which  has  combined  with  the  chlorin  of 
the  urine  to  form  silver  chlorid.  This  figure  multiplied  by  5  or 
by  8.23  gives  the  chlorin  or  sodium  chlorid  (in  milligrams)  pres- 
ent in  10  c.c.  of  urine. 

Calculate  the  twenty-four  hour  quantity. 

129 


7T  23.  Simplified  Chlorid  Determination. — The  removal  of  the 
silver  chlorid  precipitate  in  the  preceding  method  is  generally 
conceded  to  be  necessary,  because  of  the  fact  that  a  part  of  the 
silver  chlorid  is  converted  into  silver  sulphocyanate  during  the 
titration,  unless  thus  removed.  The  error  due  to  this  side  reac- 
tion when  the  chlorid  is  not  removed  is  a  loss  of  about  0.05  c.c. 
of  sulphocyanate  for  5  c.c.  of  urine,  a  loss  amounting  to  a 
deficit  of  about  o.i  g.  of  sodium  chlorid  per  liter  of  urine. 

Standard  silver  nitrate.  Dissolve  7.28  g.  of  silver  nitrate  and 
dilute  to  a  volume  of  250  c.c.  i  c.c.  is  equivalent  to  10  mg.  of 
NaCl. 

INDICATOR. — To  100  g.  of  ferric  ammonium  sulphate  add  100 
c.c.  of  water  and  200  c.c.  of  concentrated  nitric  acid.  5  c.c.  of 
the  resulting  solution  is  taken  for  each  titration. 

STANDARD  AMMONIUM  SULPHOCYANATE. — Dissolve  2  g.  of 
ammonium  sulphocyanate  in  200  c.c.  of  water  and  mix.  Transfer 
10  c.c.  of  the  standard  silver  nitrate  solution  to  a  beaker,  add 
20  c.c.  of  distilled  water  and  5  c.c.  of  the  indicator.  Fill  a  buret 
with  the  sulphocyanate  solution  and  titrate  with  constant  stirring 
until  the  characteristic  reddish  end  point  is  reached.  On  the 
basis  of  the  figure  obtained  prepare  200  or  250  c.c.  of  ammonium 
sulphocyanate  solution,  which  is  equivalent  to  the  standard  silver 
nitrate  solution.  With  a  pipet  transfer  5  c.c.  of  urine  to  a  beaker, 
add  20  c.c.  of  water,  5  c.c.  of  indicator  and  finally  (with  a  pipet) 
10  c.c.  of  silver  nitrate  solution.  While  stirring  with  a  glass  rod, 
titrate  the  surplus  silver  with  the  standard  sulphocyanate  solu- 
tion until  the  first  faint  but  unmistakable  brown  or  reddish  color- 
ation is  obtained.  On  standing  or  continued  stirring  the  color 
would  disappear,  so  the  very  first  end  point  must  be  taken. 

Subtract  the  ammonium  sulphocyanate  used  (in  c.c.)  from  10 
and  multiply  by  10.  This  gives,  in  mg.,  the  amount  of  sodium 
chlorid  present  in  5  c.c.  of  urine.  Calculate  the  twenty-four 
hour  quantity. 

24.  Indican. — To  10  c.c.  of  urine  add  2  c.c»  of  copper  sulphate 
solution,  5  c.c.  chloroform,  and  an  equal  volume  (12  c.c.)  of 
strong  hydrochloric  acid.  Close  the  mouth  of  the  tube  with  the 
thumb,  and  cautiously  invert  a  few  times. 

The  amount  of  indican  present  is  proportional  to  the  depth  of 
color  of  the  chloroform  extract.  This  qualitative  test  is  often 

131 


made  roughly  quantitative  by  using  the  color  of  Fehling's  solu- 
tion as  a  standard. 


25.  Metabolism  Experiments.  —  Weigh  accurately  a  small, 
clean,  dry  flask.  Pipet  into  it  25  c.c.  of  urine  and  weigh  again. 
From  the  data  obtained  calculate  the  specific  gravity  of  the  urine. 

Determine  the  specific  gravity  of  the  same  sample  of  urine 
by  means  of  an  ordinary  clinical  areometer.  Compare  the  results 
obtained,  and  explain  how  to  standardize  a  clinical  areometer. 

Collect  a  full  twenty-four  hour  quantity  of  urine,  and  in  it  de- 
termine the  following:  Volume,  specific  gravity,  acidity,  total 
nitrogen,  urea,  ammonia,  uric  acid,  creatinin,  chlorids,  phosphates, 
sulphates,  ethereal  sulphates.  Test  qualitatively  for  indican. 

For  two  days  eat  no  meat,  fish,  eggs,  milk,  cheese,  peas,  or 
beans,  and  only  a  little  bread.  Eat  much  butter,  potatoes,  vege- 
tables, starch  preparations,  fruit,  and  candy.  Repeat  all  the  de- 
terminations with  the  second  twenty-four  hour  quantity. 

For  two  days  eat  all  the  meat  products  you  can,  and  collect  the 
two  twenty-four  hour  quantities  of  urine.  In  the  second  twenty- 
four  hour  quantity  determine  all  the  factors  enumerated  above. 
Tabulate  and  compare  the  results  obtained  in  the  three  series  of 
analyses. 

Take  15  g.  of  sodium  bicarbonate  in  divided  doses,  collect  the 
twenty-four  hour  urine,  and  estimate  the  ammonia  and  acidity. 
Compare  with  the  normal. 

Two  days  later,  beginning  in  the  morning,  take  5  g.  ammonium 
chlorid  in  the  course  of  the  day.  Determine  the  ammonia  and 
acidity. 

Eat  much  sweetbread,  kidney,  or  liver,  for  one  day  ;  collect  the 
urine  for  the  whole  twenty-four  hours,  and  estimate  the  uric  acid. 
Compare  with  the  uric  acid  figures  previously  obtained.  Explain. 


133 


PAUT  VII 
BLOOD 

1.  Hemoglobin  Crystals.— Place   a   drop   of   defibrinated   rat 
blood  on  a  slide,  add  a  drop  or  two  of  water,  mix,  and  cover 
with  a  cover-glass.     Sketch  the  crystals  which  separate  after  a 
few  minutes. 

2.  Hemoglobin  (Reduced  Hemoglobin). — Add  to  dilute  blood 
a  few  drops  of  strong  ammonium  sulphid,  or  one  or  two  drops 
of  freshly  prepared  Stokes'  reagent. 

Examine  spectroscopically. 

Stokes'  reagent  is  a  2  per  cent,  solution  of  ferrous  ammonium 
sulphate  in  3  per  cent,  tartaric  acid,  to  which  is  added  ammonia 
until  a  clear  solution  is  obtained.  The  ammonia  should  be  added 
only  to  the  amount  of  reagent  immediately  needed. 

Shake  the  solution  of  hemoglobin  with  air,  and  note  the  rapid 
change  to  oxyhemoglobin.  Change  the  same  solution  of  oxyhemo- 
globin  to  hemoglobin,  and  reverse  two  or  three  times,  and  note 
the  facility  with  which  hemoglobin  takes  up  and  loses  oxygen. 

3.  Carbon  monoxid  hemoglobin. — Pass  a  current  of  illuminat- 
ing gas  through  a  dilute  oxyhemoglobin  solution  for  a  minute, 
and  filter.    Note  the  change  of  color.    Try  the  effect  on  the  solu- 
tion of  (i)  ammonium  sulphid,  (2)  Stokes'  reagent,  (3)  potas- 
sium ferrocyanid,   (4)   shaking  with  air.     Note  the  stability  of 
the  compound. 

Examine  spectroscopically. 

4.  Methemoglobin. — Add  to  dilute  defibrinated  blood  (1:15) 
two  drops  of  a  freshly  prepared  solution  of  sodium  nitrite.    Note 
the  change.    What  is  the  effect  produced  by  the  addition  of  re- 
ducing agents? 

135 


5.  Hematin. — Hemolyze  a  small  quantity  of  blood  and  add 
dilute  hydrochloric  acid  cautiously  till  a  precipitate  occurs. 

Acidify  strongly  with  hydrochloric  acid.  Note  color  (acid 
hematin).  Then  add  sodium  hydrate  till  strongly  alkaline.  Note 
color  (alkaline  hematin).  To  the  alkaline  solution  add  a  few 
drops  ammonium  sulphid  and  warm  gently.  Note  color  (reduced 
hematin  or  hemochromogen) . 

6.  Hemin  Crystals — Teishmann's  Test. — Place  a  bit  of  pow- 
dered dried  blood  on  a  glass  slide,  add  a  minute  crystal  of  sodium 
iodid  and  two  drops  of  glacial  acetic  acid.    Cover  with  a  cover- 
glass  and  warm  gently  over  a  flame  until  bubbles  appear.     De- 
scribe the  crystals  which  separate. 

7.  Fibrinogen. — Allow  about  6-8  volumes  fresh  blood  to  run 
from  the  animal  into  i  volume  of  a  I  per  cent,  potassium  oxalate 
solution  (why?).    Allow  to  stand  over  night  in  the  cold  room,  and 
siphon  off  the  clear  plasma.    With  the  solution  so  obtained  make 
the  following  experiments: 

Dilute  10  c.c.  with  20  c.c.  of  distilled  water  and  divide  into  3 
equal  portions.  To  one  add  a  little  dilute  (i  per  cent.)  calcium 
chlorid  solution.  To  the  second  add  a  few  drops  of  blood  serum 
(why?).  Place  the  three  tubes  in  a  beaker  of  water  heated  to 
40°  and  observe  the  time  of  clotting. 


137 


PART  VIII 
MILK 

Determine  the  specific  gravity  as  in  the  case  of  urine. 

1.  Determination  of  Total  Nitrogen. — Transfer  25  c.c.  milk  to 
a  100  c.c.  volumetric  flask.    Fill  to  the  mark  with  water,  mix,  and 
determine  the  total  nitrogen  in  i  c.c.    Calculate  the  total  protein 
content  of  the  milk  by  multiplying  its  nitrogen  with  the  fac- 
tors 6.25. 

2.  Determination  of  Casein.— Transfer  50  c.c.  of  the  diluted 
milk  to  another  100  c.c.  flask,  and  carefully  precipitate  the  casein 
by  the  addition  of  dilute  acetic  acid  and  gentle  shaking.     Make 
up  to  volume  (100  c.c.)  with  water.     Centrifuge  a  portion,  and 
determine  the  nitrogen  in  5  c.c.  of  the  clear  liquid. 

Calculate  the  total  nitrogen  (and  protein),  making  due  allow- 
ance for  the  dilutions,  and  subtract  from  the  total  protein  found 
in  the  preceding  experiment.  The  difference  represents  casein. 

3.  Determination  of  Milk  Sugar. — Transfer  5  c.c.  of  milk  to 
a  small  flask  or  beaker.     Add  20  c.c.  of  water  and  mix  well. 
Fill  the  special  5   c.c.  sugar  buret,  used  in  glucose  titrations, 
with  the  diluted  milk.     Transfer  to  a  large  test  tube  5  c.c.  of 
5.9  per  cent,  copper  sulphate  solution  and  i  c.c.  of  saturated  sodic 
carbonate  solution.    Shake  for  a  moment,  then  add  about  5  g.  of 
the  salt  mixture  used  in  glucose  titrations.     (p.  57.)     Add  4.2 
c.c.  of  diluted  cow's  milk  or  2.8  c.c.  of  diluted  mother's  milk  and 
boil  gently  for  four  minutes  counting  from  the  time  of  actual 
beginning  boiling.     At  the  end  of  four  minutes  add  more  milk 
(0.02  to  0.3  c.c.)  unless  it  is  apparent  that  the  initial  addition  is 
enough.     Boil  one  minute  after  each  fresh  addition. 

In  the  sugar  titration  in  the  case  of  milk  one  can  assume  that 
cow's  milk  will  contain  a  little  less  than  5  per  cent,  and  that 
mother's  milk  may  contain  no  more,  but  may  go  as  high  as  7  per 

139 


cent.,  hence  with  mother's  milk  it  is  not  advisable  to  start  with 
more  than  2.8  c.c.  for  the  first  boiling  period. 

Calculation:  5  c.c.  of  the  copper  sulphate  solution  is  reduced 
in  about  5  minutes  by  40.4  mg.  of  lactose.  4.04  times  5,  the 
degree  of  dilution,  or  20.2,  divided  by  the  volume  of  diluted 
milk  used  gives  the  per  cent,  of  lactose  in  the  milk. 

4.  Determination  of  Fat. — Measure  out  17.6  c.c.  of  thoroughly 
mixed  milk  into  a  Babcock  nask.  Add  17  c.c.  sulphuric  acid  (sp. 
gr.  1.82)  and  mix  thoroughly,  with  gentle  turning  and  shaking, 
until  all  the  precipitated  proteins  have  dissolved.  Rotate  in  the 
centrifuge  for  3  minutes.  Add  hot  water  up  to  the  beginning 
of  the  graduations  in  the  neck  of  the  flask,  and  rotate  for  I  min- 
ute. The  graduations  read  in  per  cent,  of  fat. 


141 


PART  IX 
BONE 

Weigh  a  piece  of  clean,  raw  bone  on  the  laboratory  scales.  Im- 
merse in  about  10  times  its  weight  of  10  per  cent,  hydrochloric 
acid  in  a  flask.  If  any  gas  is  evolved,  determine  what  it  is. 

After  48  hours  dilute  the  volume  of  the  solution  and  what  re- 
mains of  the  bone  to  a  definite  volume  in  a  cylinder.  Mix  so  as 
to  get  the  solution  uniform  in  composition. 

Pipet  out  25  c.c.  of  the  solution,  neutralize  with  sodic  hydrate, 
using  congo  red  paper  as  indicator,  and  determine  the  phosphates. 
Repeat. 

Calculate  the  tricalcic  phosphate  corresponding  to  the  phos- 
phoric acid  found. 

Taking  other  portions  of  the  original  solution,  demonstrate  ex- 
perimentally that  all  the  calcium  in  the  bone  can  not  be  precipi- 
tated together  with  the  phosphoric  acid  present. 

In  what  form  is  this  excess  of  calcium  present  in  bone? 

Examine  the  insoluble  substance  left  in  the  hydrochloric  acid 
solution.  What  is  the  substance?  Prepare  a  "gelatin"  solution 
from  it. 


143 


PART  X 
BILE 

1.  Character  of  Bile. — Determine  the  specific  gravity,  taste, 
odor,  color,  consistency,  reaction,  of  the  bile  supplied. 

Test  for  coagulable  protein. 

2.  Bile  Salts. — Mix  250  c.c.  ox-bile  with  one-fourth  its  volume 
of  bone-black,  and  evaporate  nearly  to  dryness  on  the  water-bath. 
Cool,  transfer  the  residue  to  a  flask,  and  extract  with  200  c.c.  of 
alcohol  over  night.     Filter,  and  evaporate  the  filtrate  to  dryness 
on  the  water-bath.    Dissolve  the  residue  in  absolute  alcohol,  and 
filter  into  a  dry  flask.    Add  anhydrous  ether  till  permanent  cloudi- 
ness develops.    Place  in  the  cold  room  to  crystallize.    Filter.    De- 
scribe the  crystals. 

3.  Pettenkofer's  Test  for  Bile  Salts.— Mix  a  little  bile  with  2 
or  3  drops  of  10  per  cent,  solution  of  cane  sugar.    Place  in  a  test 
tube  some  concentrated  sulphuric  acid.    Incline  the  tube  contain- 
ing the  sulphuric  acid,  and  pour  the  bile  solution  slowly  down 
the  side  of  the  tube  so  that -it  forms  a  layer  above  the  sulphuric 
acid. 

4.  G-melin's  Test  for  Bile  Pigments. — Put  5  c.c.  of  nitric  acid, 
containing  some  nitrous  acid,  in  a  test  tube,  and  introduce  on  top 
of  it   (pipet)   about  5  c.c.  of  diluted  bile.     Note  what  occurs. 
Study  the  delicacy  of  the  reaction  with  very  dilute  solution  of 
bile. 

5.  Test  for  Bile  in  Urine. — The   presence   of   bile   in   human 
urine  is  usually  indicated  by  its  color  and  the  color  of  the  foam. 
In  making  the  nitric  acid  test  for  albumin  the  presence  of  bile  is 
also  revealed  by  a  series  of  colored  rings   (green,  blue,  violet, 
red,  and  yellowish-red). 

145 


A  similar  series  of  colors  is  occasionally  obtained  from  urines 
which  have  been  preserved  with  thymol.  This  is  one  of  the  objec- 
tions to  this  otherwise  excellent  preservative. 

To  10  c.c.  of  urine  add  a  few  drops  of  calcium  chlorid  solution 
and  a  few  drops  of  10  per  cent,  sodic  hydrate.  Filter.  Remove 
the  filter  paper  from  the  funnel,  open  it,  and  drop  I  or  2  drops 
of  concentrated  nitric  acid  on  the  sediment.  In  the  presence  of 
(human)  bile  the  usual,  characteristic  series  of  colored  rings  is 
obtained. 


147 


SUPPLEMENT 


URINE 

Qualitative  Test  for  Acetone  in  Urine. — Clinicians  seldom  dif- 
ferentiate between  acetone  and  diacetic  acid,  and  the  "acetone 
tests"  which  they  use  are  tests  for  diacetic  acid  rather  than  for 
acetone.  (See  p.  159.) 

In  the  qualitative  test  for  acetone,  as  for  its  quantitative  de- 
termination, the  acetone  is  first  removed  from  the  urine  by  means 
of  an  air  current,  just  as  in  corresponding  determinations  (and 
tests)  for  ammonia. 

In  the  large  test  tube  used  for  the  colorimetric  determination 
of  ammonia  place  first  5  c.c.  of  urine  and  1-2  drops  dilute  acid 
(HC1  or  H2SO4).  Then  insert  the  rubber  stopper  carrying  the 
absorption  tube,  etc.,  place  the  test  tube  in  a  beaker  of  lukewarm 
water  (35-40°  C),  and  aspirate  the  volatile  acetone  by  means  of 
a  moderately  rapid  air  current  into  a  test  tube  containing  5  c.c. 
distilled  water  and  5  c.c.  Scott-Wilson  reagent.  If  acetone  is 
present,  even  if  only  in  minute  traces,  the  solution  becomes  tur- 
bid. If  the  amount  of  acetone  obtained  is  extremely  small  the 
turbidity  may  not  appear  for  5-10  minutes. 

The  Scott-Wilson  reagent  for  acetone,  which  is  used  for  qualita- 
tive tests  as  well  as  for  quantitative  determinations,  is  most  con- 
veniently prepared  as  follows: 

To  10  g.  of  mercuric  cyanid  dissolved  in  600  c.c.  of  water  add  a 
cooled  solution  of  180  g.  of  sodium  hydroxid  in  600  c.c.  of  water. 
Transfer  this  mixture  to  a  heavy  walled  glass  jar,  and  to  it  add  2.9 
g.  of  silver  nitrate  dissolved  in  400  c.c.  of  water.  The  silver  solution 
should  be  added  in  a  slow  stream,  and  the  addition  must  be  accom- 
panied by  constant  and  exceedingly  vigorous  stirring  with  a  heavy 
glass  rod.  If  properly  made,  the  silver  dissolves  completely,  giving 
a  clear  solution  which  is  at  once  available  for  use.  If  the  solution 
is  turbid,  it  should  be  set  aside  to  settle  for  three  or  four  days  and 
the  clear  supernatant  liquid  removed  by  means  of  a  siphon. 

In  the  clear  reagent  a  new  sediment  gradually  forms,  so  that  the 

151 


solution  deteriorates  slowly  and  after  a  few  months  is  not  service- 
able for  quantitative  work,  though  still  good  for  qualitative  tests. 

Titration  of  Acetone  and  Preparation  of  Standard  Acetone  Solu- 
tions.— Standard  solutions  of  iodin,  sodium  thiosulphate,  and  potas- 
sium permanganate  are  needed  in  this  work,  the  latter  being  used 
only  as  a  basis  for  making  the  other  two  accurate.  .5  N  perman- 
ganate solution  may  be  used. 

Iodin — Weigh  roughly  10-12  g.  of  potassium  iodid  in  a  beaker  and 
add  50  c.c.  of  water.  Weigh  out  6.4  g.  iodin  in  a  small  beaker  cov- 
ered with  a  watch  glass  and  add  this  to  the  potassium  iodid  solution. 
Stir  until  the  iodin  is  dissolved  and  then  transfer  the  resulting  solu- 
tion to  a  500  c.c.  volumetric  flask.  Dilute  to  the  mark  with  water 
and  mix. 

Sodium  Thiosulphate,  Weigh  out  24.85  g.  of  the  salt  (Na2S2O3-|- 
5H2O),  dissolve  in  water,  transfer  to  a  500  c.c.  volumetric  flask,  fill 
to  the  mark  with  water,  and  mix. 

The  two  solutions  thus  prepared  should  be  approximately  tenth 
normal.  Their  relative  values  are  determined  by  titration  as  fol- 
lows: 

Pipet  20  c.c.  of  the  iodin  solution  into  a  flask  (capacity  500-600 
c.c.)  and  add  about  100  c.c.  water.  From  a  buret,  run  in  the  thio- 
sulphate solution  until  the  reddish-brown  iodin  color  has  faded  to  a 
faint  straw  yellow.  Now  add  a  few  drops  of  starch  solution  (see 
p.  65)  and  continue  the  titration  till  the  blue  iodid  of  starch  color 
disappears.  The  end  point  of  this  titration  is  very  sharp. 

The  value  of  the  thiosulphate  solution  is  now  determined  as  fol- 
lows: 

Weigh  roughly  2  g.  potassium  iodid,  transfer  to  a  flask,  and  dis- 
solve in  about  150  c.c.  water.  Add  5  c.c.  diluted  hydrochloric  acid 
(1-5)  and  50  c.c.  .05  N  potassium  permanganate  solution. 

The  permanganate  sets  free  an  equivalent  quantity  of  iodin  accord- 
ing to  the  following  equation: 

2KMnO4  +  loKI  +  i6HCl  =  I2KC1  +  2MnCl2  +  8H2O  +  5l2 

The  iodin  thus  liberated  is  then  titrated  with  the  sodium  thiosul- 
phate solution  in  the  same  manner  as  the  original  iodin  solution. 

From  the  relative  values  of  the  iodin,  the  thiosulphate,  and  the  per- 
manganate solutions,  the  exact  values  of  the  first  two  (in  terms  of 
tenth  normal  solutions)  are  calculated. 

Standard  Stock  Solution  of  Acetone.— Add  about  i  c.c.  of  pure 
acetone  (from  the  bisulphite  compound)  to  water  in  a  one  liter  volu- 
metric flask,  dilute  to  the  mark,  and  mix.  The  titration  of  acetone 
with  iodin  is  based  on  the  fact  that  in  alkaline  solutions  the  acetone 

153 


is  converted  into  iodoform.    Several  reactions  are  involved  in  this 
process : 

Iodine  is  converted  into  hypo-iodite, 

1.  I2  +  2KOH  =  KOI  +  KI  +  H2O. 

Hypo-iodite  is  then  slowly  converted  into   (useless)   iodate, 

2.  3KOI  =  KIOa  +  2KI. 

The  hypo-iodite  converts  acetone  into  iodoform  and  acetic  acid, 

3.  3KOI  +  CHXCOCH,  =  CH,COCIa  +  sKOH. 

4.  CH3COCI3  +  KOH  =  CHSCOOK  +  CHI, 

On  acidifying,  after  the  iodoform  has  been  formed,  the  surplus 
iodin  present  as  hypo-iodite  (and  iodate)  is  set  free,  and  can  then  be 
titrated  with  the  standard  sodium  thiosulphate  solution  as  described 
above. 

Each  molecule  of  acetone  uses  up  three  molecules  of  hypo-iodite, 
and  as  each  molecule  of  the  latter  is  formed  at  the  expense  of  two 
atoms  of  iodin,  six  atoms  of  iodin  correspond  to  one  molecule  of 
acetone.  .'  One  c.c.  of  .1  N  iodin  solution  corresponds  therefore  to 
.968  mg.  acetone.  Because  of  the  iodate  formation  a  considerable 
excess  must  be  added. 

The  titration  of  the  acetone  solution  is  carried  out  as  follows : 

Transfer  25  c.c.  of  the  stock  acetone  solution  to  a  flask,  add  150- 
200  c.c.  water,  then  50  c.c.  of  the  standardized  iodin  solution,  and  10 
c.c.  strong  sodic  hydrate  (40  per  cent.).  Let  stand  with  occasional 
shaking  for  5  minutes.  Add  18  c.c.  concentrated  hydrochloric  acid, 
and  titrate  the  liberated  excess  of  iodin  with  the  standard  thiosul- 
phate solution. 

If  the  standard  solutions  are  exactly  tenth  normal,  subtract  the 
volume  of  thiosulphate  employed  from  the  volume  of  iodin  solution 
taken,  and  multiply  the  remainder  (in  c.c.)  with  .968  to  obtain  the 
acetone  content  (in  mg.). 

Calculate  the  acetone  content  of  the  stock  solution  (in  mg.  per 
c.c.).  Transfer  to  a  distilling  flask  as  much  of  it  as  contains  exactly 
50  mg.  of  acetone.  Add  water  enough  to  make  a  volume  of  500-600 
c.c.,  and  distill  with  vigorous  cooling  of  the  condenser.  The  receiver 
should  be  a  large  flask  (750-1000  c.c.)  containing  about  250  c.c. 
approximately  normal  sulphuric  acid.  Boil  for  20-30  minutes,  or 
until  at  least  150  c.c.  of  distillate  has  gone  over.  Transfer  this  dis- 
tillate to  a  volumetric  (liter)  flask  and  dilute  to  the  mark  with 
water.  Ten  c.c.  of  the  acetone  solution  so  obtained  contains  .5  mg. 
acetone.  This  solution,  as  well  as  the  original  stock  solution,  should 
be  kept  in  a  well  stoppered  bottle.  The  sulphuric  acid  present  in  the 
dilute  standard  acetone  solution  is  added  to  prevent  polymerization. 

Preparation  of  Standard  Acetone  Solutions  from  the  Acetone  Bi- 
sulphite Compound— A  standard  acetone  solution  can  be  prepared 

155 


without  distillations  from  the  "acetone  sulphite"  used  in  photography 
as  follows: 

Transfer  2.5  g.  of  the  powder  to  a  volumetric  (1,000  c.c.)  flask  by 
means  of  a  little  water  (50  c.c.),  and  fill  up  to  the  mark  with  dilute 
(i  in  5)  hydrochloric  acid.  Transfer  25  c.c.  of  the  solution  to  a 
flask.  Add  20  c.c.  tenth  normal  iodin,  let  stand  for  five  minutes,  and 
titrate  the  surplus  iodin  with  tenth  normal  thiosulphate  solution.  This 
titration  gives  the  SO2  or  the  sodium  bisulphite  content. 

To  another  25  c.c.  of  the  acetone  solution  add  50  c.c.  tenth  normal 
iodin,  let  stand  five  minutes,  then  add  10  c.c.  strong  sodic  hydrate, 
followed  after  five  minutes  by  18  c.c.  concentrated  hydrochloric  acid. 
Titrate  the  liberated  iodin  with  thiosulphate.  From  the  50  c.c.  of 
iodin  taken  subtract  (a)  the  figure  of  the  last  thiosulphate  titration 
and  (b)  the  iodin  corresponding  to  the  SO  .  The  remainder  cor- 
responds to  the  acetone.  From  the  standardized  stock  solution  pre- 
pare the  more  dilute  standard  solution  (5  c.c.  or  10  c.c.  of  which 
.should  contain  exactly  half  a  mg.  of  acetone). 

If  the  "acetone  sulphite"  is  not  available,  acetone  sodium  bisulphite 
is  easily  prepared  by  slowly  adding  (with  stirring)  two-thirds  volume 
of  ordinary  acetone  to  one  volume  (100  or  200  c.c.)  of  saturated 
sodium  bisulphite  solution  (freshly  prepared  and  filtered).  The  pre- 
cipitate should  be  freed  as  completely  as  possible  from  the  mother 
liquor  by  filtering  on  a  Buchner  funnel  with  suction.  Then  wash 
rapidly  two  or  three  times  with  alcohol.  Let  dry  in  the  open  air 
for  two  or  three  days.  Sieve  to  make  the  preparation  uniform,  and 
preserve  in  glass  stoppered  vessel. 

Quantitative  Determination  of  Acetone  in  Urine. — To  about  I 
c.c.  of  10  per  cent,  sulphuric  acid  in  a  large  test  tube  add  enough 
urine  (.5  to  5  c.c.)  to  give  about  .5  mg.  of  free  acetone  (-S-.7 
mg.).  Connect  the  test  tube,  as  in  ammonia  determinations,  with 
a  second  test  tube  containing  10  c.c.  of  fresh  approximately  2 
per  cent,  sodium  bisulphite  solution.  )Varm  the  first  test  tube  to 
35-40°  C,  as  in  the  qualitative  test  for  acetone  and  aspirate  the 
acetone  into  the  bisulphite  solution  by  means  of  a  moderate  air 
current  (time  about  10  minutes).  Transfer  the  sulphite-acetone 
solution  to  a  100  c.c.  volumetric  flask  together  with  distilled  water 
enough  to  make  50-60  c.c.  To  each  of  two  other  100  c.c.  flasks 
add  10  c.c.  of  the  standard  acetone  solution  containing  .5  mg. 
acetone,  add  10  c.c.  of  the  2  per  cent,  bisulphite  solution,  and 
dilute  with  distilled  water  to  50-60  c.c. 

To  each  of  the  three  flasks  add  15  c.c.  (clear)  Scott- Wilson  re- 
agent, and  immediately  (before  turbidity  formation)  dilute  with 

157 


distilled  water  to  the  mark,  mix,  and  let  stand  for  12-15  minutes. 
Read  the  turbid  contents  of  one  of  the  standard  acetone  suspen- 
sions against  itself  in  the  Duboscq  colorimeter.  The  best  source 
of  light  for  these  comparisons  is  diffuse  daylight  coming  through 
an  opening  (about  25  cm.  square)  cut  through  the  shade  of  a 
(north  side)  window.  The  colorimeter  metal  screen  must  also  be 
used.  The  instrument  must  be  adjusted  until  20  mm.  of  the  two 
suspensions  are  equal.  Now  replace  the  contents  of  one  of  the 
colorimeter  cups  with  the  contents  of  the  second  standard  sus- 
pension, and  the  other  with  the  unknown  acetone  mercury  sus- 
pension obtained  from  the  urine,  and  make  the  turbidity  compari- 
son in  the  same  manner  as  colorimetric  comparisons — setting  the 
standard  at  20  mm. 

Twenty  multiplied  by  .5  and  divided  by  the  reading  of  the  un- 
known (in  mm.)  gives  the  acetone  content  (in  mg.)  of  the  vol- 
ume of  urine  taken  for  the  analysis. 

Qualitative  Test  for  Diacetic  Acid  (in  traces). — To  5  c.c.  of 
urine  in  a  test  tube  add  1-2  c.c.  dilute  acetic  acid  (10  per  cent.) 
and  a  small  crystal  of  sodium  nitroprussid.  Shake  a  few  times 
to  dissolve  the  salt,  then  add  an  excess  of  concentrated  ammonia 
(2-3  c.c.),  and  mix.'  A  violet  color  indicates  diacetic  acid. 

Grerhardt's  Ferric  Chlorid  Test  for  Diacetic  Acid. — This  test  is 
useful  for  showing  the  presence  in  urine  of  relatively  large 
amounts  of  diacetic  acid.  It  is  made  as  follows :  To  5  c.c.  of 
urine  in  a  test  tube  add  ferric  chlorid  solution  (10  per  cent.), 
drop  by  drop.  At  first  a  white  precipitate  of  ferric  phosphate  is 
obtained,  then,  as  the  addition  of  the  reagent  is  continued,  a  dark 
red  color  is  produced  if  diacetic  acid  is  present  (in  more  than 
traces). 

A  number  of  substances  used  as  drugs,  such  as  salicylic  acid, 
phenacetin,  etc.,  give  a  similar  reaction.  If  confusion  due  to 
such  drugs  is  to  be  suspected,  boil  the  deep  red  solution  for  2-3 
minutes.  If  the  color  is  due  to  diacetic  acid,  it  should  disappear 
during  the  boiling  and  not  reappear  on  cooling.  The  disappear- 
ance is  due  to  the  destruction  by  boiling  of  the  unstable  diacetic 
acid. 

Quantitative  Determination  of  Diacetic  Acid  (and  Acetone).— 

Acetone  urines  contain  from  two  or  three  to  nine  or  ten  times 

159 


as  much  aceto-acetic  acid  as  acetone.  In  strictly  fresh  urines  the 
latter  proportions  prevail;  but  the  older  the  urine  the  greater 
becomes  the  relative  proportion  of  acetone,  because  of  the  spon- 
taneous decomposition  of  the  aceto-acetic  acid.  Urines  giving  a 
strong  ferric  chlorid  reaction  usually  contain  more  than  .5  mg.  of 
aceto-acetic  acid  per  cubic  centimeter,  and  must  be  diluted  so  that 
an  appropriate  fraction  of  I  c.c.  (of  the  original  urine)  can  be 
taken  for  a  determination. 

The  amount  of  urine  taken  should  yield  approximately  .5  mg. 
of  acetone  (from  .3  to  .7  mg.).  Transfer  this  amount  of  urine 
to  a  large  test  tube  containing  i  c.c.  of  10  per  cent,  sulphuric 
acid,  and  connect  with  a  second  test  tube  containing  10  c.c.  of 
2  per  cent,  sodium  bisulphite  solution.  Immerse  the  test  tube 
containing  the  urine  in  a  beaker  of  boiling  water  and  the  second 
test  tube  in  cold  water,  then  pass  through  an  extremely  slow  air 
current  for  ten  minutes.  Increase  slightly  the  speed  of  the  air 
current  and  continue  the  aspiration  for  another  five  minutes. 
The  aceto-acetic  acid  plus  acetone  is  thus  transferred,  in  the  form 
of  acetone,  to  the  bisulphite  solution.  Rinse  the  solution  into  a 
100  c.c.  volumetric  flask,  and  determine  the  acetone  exactly  a?  in 
the  determination  of  the  performed  acetone. 

One  mg.  of  acetone  is  equivalent  to  1.8  mg.  of  aceto-acetic  acid. 
From  the  "total  acetone"  of  the  24-"hour  quantity  of  urine  is  sub- 
tracted the  total  preformed  acetone,  and  the  remainder  multi- 
plied by  i  .8  gives  the  aceto-acetic  acid. 

Determination  of  Beta-oxybutyric  Acid  in  Urine, — The  method 
described  below  was  at  first  thought  by  its  authors  (Folin  and 
Denis)  to  give  strictly  all  the  beta-oxybutyric  acid  present  in 
urine.  But  it  now  appears  that  the  yield  is  only  85-95  per  cent., 
just  as  in  the  original  method  of  Shaffer. 

The  urine  is  diluted  from  10-100  times,  depending  on  how  much 
of  the  substance  is  present.  The  ammonia  content  of  the  urine 
is  the  best  index  as  to  how  much  urine  is  required  to  yield  the 
desired  amount  of  beta-oxybutyric  acid  (1.5-3.5  m£-)-  The  fer- 
ric chlorid  test  for  diacetic  acid  is  also  helpful,  but  without  con- 
siderable experience  only  a  preliminary  determination  can  give 
the  desired  information. 

Measure  diluted  urine,  equivalent  to  1.5-3.5  mg.  of  beta-oxy- 
butyric acid,  into  a  500  c.c.  Kjeldahl  flask,  add  a  little  dilute  sul- 
phuric acid  (5  ctc.),  and  water  enough  to  make  a  volume  of  about 

161 


150  c.c.  Boil  the  mixture  gently  for  ten  minutes  (to  drive  off  the 
preformed  acetone  and  the  diacetic  acid),  then  add  to  the  solution 
(with  a  cylinder)  25  c.c.  of  a  solution  containing  I  per  cent,  po- 
tassium dichromate  and  35  per  cent,  sulphuric  acid,  and  connect 
the  flask,  in  the  usual  manner,  with  a  condenser  by  means  of  a 
specially  treated  rubber  stopper.  . 

The  rubber  stopper  should  be  boiled  twice  for  an  hour  in  10  per 
cent,  sodic  hydrate  solution  (or  better,  heated  in  an  autoclave  in  the 
same  solution  for  half  an  hour  at  130-140°  C.),  and  then  thoroughly 
washed.  It  is  also  necessary  to  wrap  the  stopper  thoroughly  in  tin 
foil  during  the  distillation,  so  as  to  exclude  the  volatile  sulphur  im- 
purities which  otherwise  are  given  off  and  interfere  with  the  subse- 
quent turbidity  formation. 

Distill  very  slowly,  for  one  and  one-half  hours,  collecting  the 
distillate  (about  100  c.c.)  in  another  500  c.c.  Kjeldahl  flask,  pre- 
viously charged  with  about  100  c.c.  of  water. 

To  the  distillate  add  a  small  amount  sodium  peroxide  (2  g.), 
and  redistill  by  ordinary  rapid  boiling.  Collect  this  final  distil- 
late in  a  100  c.c.  volumetric  flask  (or  cylinder).  About  80  c.c. 
should  be  obtained. 

Dilute  this  distillate  to  the  100  c.c.  mark  with  distilled  water 
and  mix.  Transfer  from  25  to  50  c.c.  into  a  100  c.c.  volumetric 
flask,  and  determine  the  acetone  content  by  the  turbidity  method, 
as  in  the  case  of  the  two  preceding  (acetone)  determinations.  No 
bisulphite  is  used  in  this  case  to  hold  the  acetone,  and  none  should 
therefore  be  added  to  the  standard.  Each  milligram  of  acetone 
obtained  corresponds  to  1.78  mg.  of  beta-oxybutyric  acid. 

Shaffer's  Short  Method  for  the  Determination  of  Beta-oxybutyric 
Acid. — To  50  c.c.  of  urine  add  100  c.c.  of  water,  then  50  c.c.  of 
basic  lead  acetate  solution  (Goulard's  Ext.  U.S. P.),  and  stir.  Add 
50  c.c.  approximately  normal  NaOH  and  stir  again.  Filter.  A 
clear  filtrate  containing  but  traces  of  lead  or  glucose  is  usually 
obtained,  even  though  the  original  urine  contained  considerable 
quantities  of  sugar.  Traces  of  sugar  do  not  interfere  with  the 
determination. 

Introduce  50  c.c.  of  the  filtrate  into  a  500  c.c.  Kjeldahl  flask 
previously  marked  at  the  level  of  100  c.c.  with  a  "glass  pencil." 
Add  25  c.c.  of  water  and  50  c.c.  of  half  concentrated  sulphuric 

163 


acid.     The   latter,   if    freshly   prepared  by  mixing   with   water 
( i :  i ) ,  must  be  cooled  before  it  is  used. 

Connect  the  Kjeldahl  flask  with  a  dropping  funnel  and  with  a 
condenser.  Distill  off  about  25  c.c.,  collecting  the  distillate  in  an- 
other Kjeldahl  flask. 

The  first  distillate  thus  obtained  contains  the  preformed  acetone, 
as  well  as  the  acetone  derived  from  the  aceto-acetic  acid  of  the  urine. 
By  adding  to  it  5  c.c.  of  strong  alkali  and  redistilling,  for  10  minutes, 
this  acetone  is  obtained,  in  the  second  distillate,  free  from  impurities, 
and  can  be  titrated  with  iodin  and  thiosulphate. 

After  replacing  the  Kjeldahl  flask  used  as  a  receiver  with  an- 
other one,  the  distillation  of  the  urine  filtrate  is  continued,  while 
adding  slowly  (about  15  drops  per  10  seconds)  a  .2  per  cent,  po- 
tassium bichromate  solution. 

During  this  distillation  the  volume  in  the  distilling  flask  should 
be  kept  at  approximately  100  c.c.  (i.e.,  at  the  level  indicated  by 
the  pencil  mark).  This  is  readily  accomplished  by  regulating  the 
speed  of  the  distillation  so  that  it  just  about  equals  the  speed  with 
which  the  bichromate  solution  is  added.  The  speed  of  the  oxi- 
dation is  much  greater  with  increasing  concentration  of  sulphuric 
acid.  With  too  great  concentration  of  the  acid,  however,  when 
the  volume  approaches  a  level  of  about  70  c.c.,  the  oxybutyric 
acid  is  in  part  converted  into  crotonic  acid,  and  thus  escapes  oxi- 
dation to  acetone. 

The  bichromate  solution  is  added  only  so  fast  as  to  maintain 
a  very  slight  excess ;  the  blue  green  color  should  largely  predomi- 
nate in  the  boiling  mixture.  Occasionally  it  may  be  necessary  to 
interrupt  the  addition  of  bichromate  for  a  few  minutes,  but  the 
volume  in  the  distilling  flask  should  not  be  allowed  to  sink  below 
85  or  90  c.c.  The  addition  of  bichromate  should  be  continued 
until  (at  the  concentration  of  acid  used)  no  more  appears  to  be 
converted  into  the  green  chromium  salt.  From  50  c.c.  to  100  c.c. 
bichromate  solution  (=  .1  g.-.2  g.  K2C2O7)  is  usually  required 
for  each  distillation.  The  addition  (and  distillation)  lasts  20-30 
minutes. 

The  distillate  obtained  must  be  redistilled,  after  the  addition 
of  5  c.c.  strong  alkali  and  about  20  c.c.  of  30  per  cent,  hydrogen 
peroxide.  This  final  distillation  need  not  last  more  than  10  min- 
utes. The  distillate  thus  obtained  is  titrated  in  the  usual  manner 
(p.  155)  with  iodin  and  thiosulphate.  The  yield  of  acetone  ob- 

165 


tained  is  about  90  per  cent,  of  the  theoretical  amount  when  work- 
ing with  solutions  of  pure  beta-oxybutyric  acid.  A  correction  of 
10  per  cent,  should  therefore  be  added  to  the  results  obtained. 

Slightly  higher  results  (93-94  per  cent.)  may  be  obtained  by 
a  very  slow  addition  of  the  bichromate,  and  a  considerable  pro- 
longation of  the  distillation  period,  but  since  the  theoretical 
amounts  of  acetone  cannot  be  obtained  the  advantage  so  gained 
is  doubtful. 

Colorimetric  Method  for  the  Determination  of  Phenols  in  Urine. 

— The  phosphotungstic  phosphomolybdic  reagent  described  in 
connection  with  the  colorimetric  determination  of  uric  acid  (p. 
205)  was  originally  devised  as  a  reagent  for  phenols,  and  is 
serviceable  for  the  determination  of  phenols  in  urinary  filtrates 
from  which  the  uric  acid  has  been  removed. 

Transfer  10  c.c.  of  ordinary,  or  20  of  very  dilute,  urine  to  a 
50  c.c.  volumetric  flask.  Add  acid  silver  lactate  solution  *  (from 
2  to  10  c.c.)  until  no  more  precipitate  is  obtained,  then  add  a  few 
drops  of  colloidal  iron,  and  shake.  Fill  to  the  mark  with  dis- 
tilled water,  shake  again,  and  filter.  By  means  of  this  precipi- 
tation uric  acid  and  traces  of  proteins  are  quantitatively  removed. 
Transfer  25  c.c.  of  the  filtrate  to  a  50  c.c.  volumetric  flask,  and 
to  it  add  a  sufficient  quantity  of  saturated  sodium  chlorid  solu- 
tion (containing  10  c.c.  of  strong  hydrochloric  acid  per  liter)  to 
precipitate  all  the  silver.  Fill  to  the  mark  with  distilled  water 
and  filter. 

To  determine  "free"  (non-conjugated)  phenols,  place  20  c.c. 
of  this  filtrate  in  a  50  c.c.  flask,  and  treat  with  5  c.c.  of  the  phos- 
photungstic phosphomolybdic  acid  reagent  and  15  c.c.  of  sat- 
urated sodium  carbonate  solution.  After  diluting  to  volume  with 
lukewarm  water  (30-35°  C.)  and  allowing  to  stand  for  twenty 
minutes,  read  the  deep  blue  solution  in  a  Duboscq  colorimeter 
against  a  standard  solution  of  phenol. 

To  determine  total  (free  and  conjugated)  phenols,  transfer 
20  c.c.  of  the  same  filtrate  to  a  large  test  tube,  add  ten  drops  of 
concentrated  hydrochloric  acid,  and  cover  the  test  tube  with  a 
small  funnel.  Heat  rapidly  to  boiling  over  a  free  flame,  and  then 
place  in  a  boiling  water-bath  (usually  a  tall  beaker)  for  ten  min- 
utes. At  the  end  of  this  time  remove  the  tube,  cool,  and  trans- 

*  This  solution  consists  of'  a  5  per  cent,  silver  lactate  solution  in  5  per 
cent,  lactic  acid. 

167 


fer  the  contents  to  a  100  c.c.  volumetric  flask.  Add  10  c.c.  of  the 
phosphotungstic  phosphomolybdic  reagent  and  25  c.c.  of  sat- 
urated sodium  carbonate  solution.  Make  up  to  volume,  shake, 
and  let  stand  for  20  minutes.  Read  against  a  standard  solution 
of  phenol. 

The  standard  is  a  solution  of  pure  phenol  in  .01  N  HC1,  con- 
taining .5  mg.  of  the  former  substance  in  5  c.c.  To  5  c.c.  of 
the  standard  solution  in  a  100  c.c.  flask  add  10  c.c.  of  the  reagent 
and  25  c.c.  of  saturated  sodium  carbonate  solution.  Fill  up  to 
the  mark  with  water  (at  about  30°  C),  and  make  the  .color  com- 
parison in  the  usual  manner,  setting  the  standard  at  20  mm. 
As  phenol  is  an  exceedingly  hygroscopic  substance,  it  is  neces- 
sary to  standardize  the  solution  by  means  of  the  iodometric  ti- 
tration. 

This  titration  is  carried  out  as  follows:  Make  a  phenol  solution 
in  .1  N  HC1,  containing  i  mg.  of  crystallized  phenol  per  c.c.  Trans- 
fer 25  c.c.  of  the  phenol  solution  to  a  250  c.c.  flask,  add  50  c.c.  .1  N 
sodic  hydrate,  heat  to  65°  C.,  add  25  c.c.  .1  N  iodin  solution,  stopper 
the  flask,  and  let  stand  at  room  temperature  thirty  to  forty  minutes. 
Add  5  c.c.  of  concentrated  hydrochloric  acid  and  titrate  excess  of 
iodin  with  .1  N  sodium  thiosulphate  solution.  One  c.c.  of  .1  N  iodin 
solution  corresponds-  to  1.567  mg.  of  phenol.  On  the  basis  of  the 
results,  dilute  the  phenol  solution  so  that  10  c.c.  contains  I  mg.  of 
phenol. 

Because  of  the  red  precipitate  in  the  solution  it  is  rather  difficult 
to  see  the  end  point  of  the  titration.  For  those  who  have  not  had 
much  experience  it  may  be  advisable  to  dilute  the  solution  to  a  defi- 
nite volume  (after  adding  the  hydrochloric  acid),  then  to  filter,  and 
to  titrate  a  portion  of  the  filtrate  as  recommended  by  Sutton;  with  a 
little  practice,  however,  the  titration  can  be  made  without  this  pro- 
cedure. 

Quantitative  Determination  of  Hippuric  Acid  in  Urine.  —In  this 
method  the  hippuric  acid  is  first  hydrolyzed  and  the  resulting  ben- 
zoic  acid  is  extracted  with  chloroform  and  the  chloroform  solu- 
tion is  titrated  with  standard  alcoholic  sodic  hydrate. 

Transfer  100  c.c.  of  urine  to  an  evaporating  dish,  add  10  c.c. 
5  per  cent,  sodic  hydrate  solution,  and  evaporate  to  dryness  on 
the  water-bath.  Rinse  the  residue  into  a  500  c.c.  Kjeldahl  flask 
by  means  of  25  c.c.  of  water  and  25  c.c.  concentrated  nitric  acid. 
Add  .2  g.  copper  nitrate,  a  couple  of  pebbles  to  prevent  bumping, 
and  boil  very  gently  over  a  microburner  for  four  and  one-half 

169 


hours.  During  this  boiling  a  miniature  Hopkins'  condenser 
(made  from  a  large  test  tube)  is  kept  within  the  neck  of  the 
boiling  flask  to  prevent  loss  of  benzoic  acid  which  is  volatile 
with  steam. 

After  cooling  rinse  the  condenser  with  25  c.c.  water,  and  trans- 
fer the  contents  of  the  flask  to  a  separatory  funnel  (capacity  500 
c.c.).  Rinse  the  flask  with  25  c.c.  water,  thus  making  the  total 
volume  in  the  separatory  funnel  100  c.c. 

Add  to  this  solution  55  g.  of  ammonium  sulphate,  shake  until 
dissolved,  and  extract  with  neutral  (freshly  washed)  chloroform 
four  times,  using  50,  35,  25,  and  25  c.c.  of  chloroform  respec- 
tively. Collect  the  chloroform  extracts  in  another  separatory 
funnel,  and  wash  this  by  shaking  with  100  c.c.  saturated  solu- 
tion of  pure  sodium  chlorid,  to  each  liter  of  which  has  been  added 
.5  c.c.  concentrated  hydrochloric  acid. 

Draw  off  the  chloroform  which  contains  the  benzoic  acid  into 
a  dry  flask,  and  titrate  with  a  dilute  standardized  sodium  alco- 
holate  solution  and  4-5  drops  of  phenolphthalein  as  indicator. 
The  first  distinct  coloration  diffusing  through  the  whole  liquid 
is  taken  as  the  end  point  without  regard  to  subsequent  fad- 
ing. 

The  sodium  ethylate  solution  is  made  by  dissolving  from  1.8  g. 
to  2.3  g.  metallic  sodium  in  absolute  alcohol  and  diluting  to  a  liter 
with  absolute  alcohol.  It  is  standardized  against  chloroform 
solutions  of  benzoic  acid. 

One  cubic  centimeter  of  .1  N  alcoholate  corresponds  to  1.22 
mg.  benzoic  acid  or  1.79  mg.  hippuric  acid. 

Turbidity  Method  for  the  Determination  of  Albumin  in  Urine. — 

To  about  75  c.c.  of  water  in  each  of  'two  100  c.c.  volumetric  flasks 
add  5  c.c.  of  a  25  per  cent,  solution  of  sulphosalicylic  acid.  To 
one  flask  add  5  c.c.  of  a  standard  protein  solution,  prepared  as 
described  below,  and  containing  10  mg.  of  albumin.  To  the  other 
add  the  albuminous  urine  I  c.c.  at  a  time  (by  means  of  an  Ost- 
wald  pipet)  until  the  turbidity  obtained  seems  to  be  reasonably 
near  that  of  the  standard.  Fill  the  two  flasks  up  to  the  mark 
with  water,  cautiously  inverting  a  few  times  to  secure  mixing. 
The  standard  must  invariably  first  be  read  against  itself  to  secure 
the  adjustment  of  the  colorimeter  (and  of  the  eye).  Then  re- 
place the  contents  of  one  of  the  Duboscq  colorimeter  cups  by 

171 


the  suspension  of  the  unknown,  and  make  the  turbidity  compari- 
son in  the  usual  manner. 

Set  the  standard  containing  10  mg.  of  protein  at  20  mm.  The 
unknown  must  not  read  less  than  10  nor  more  than  30  mm.  Di- 
viding 200  by  the  product  of  the  reading  of  the  unknown  and 
the  number  of  cubic  centimeters  of  urine  taken,  gives  the  albumin 
in  milligrams  per  cubic  centimeter  of  urine. 

It  is  very  important  not  to  shake  the  albuminous  suspensions 
in  the  volumetric  flasks  because  of  the  tendency  of  the  precipitate 
to  agglutinate.  The  preliminary  mixing  must  therefore  be  accom- 
plished by  means  of  a  few  gentle  inversions. 

The  standard  protein  solution  is  prepared  from  fresh  blood  serum 
free  from  hemoglobin.  For  the  preparation  of  this  serum  either 
slaughter  house  or  normal  human  blood  may  be  used.  The  so-called 
blood  serum  sold  for  the  preparation  of  bacteriological  culture  media 
should  be  avoided,  as  it  is  usually  several  days  old  and  is  frequently 
partially  decomposed.  The  dried  preparations  of  "blood  albumin" 
listed  by  chemical  dealers  are  also  not  satisfactory  for  the  prepara- 
tion of  standard  solutions.  To  prepare  the  standard,  dilute  25-35  c.c. 
of  serum  with  a  15  per  cent,  solution  of  chemically  pure  sodium 
chlorid  to  about  1500  c.c.  Mix  and  filter.  By  means  of  nitrogen  de- 
terminations ascertain  the  protein  content  of  the  filtrate  ( protein  — 
N  X  6.25)  and  on  the  basis  of  the  figure  obtained,  dilute  the  solution 
with  15  per  cent,  sodium  chlorid  solution  so  that  it  contains  2  mg.  of 
protein  per  cubic  centimeter.  Sodium  chlorid  in  the  concentration 
mentioned  is  fairly  effective  as  a  preservative.  Nevertheless  it  is  best 
to  saturate  the  standard  albumin  solution  with  chloroform  (20  c.c.). 

The  above  method  is  not  applicable  to  urines  which  are  very 
deeply  colored  with  blood  or  bile  pigments.  The  method  is  of 
course  applicable  to  other  albuminous  fluids  than  urine,  as,  for 
example,  exudates,  transudates,  and  the  cerebrospinal  fluid. 

Gravimetric  Method  for  the  Determination  of  Albumin  in  Urine. 
— The  method  is  as  follows:  Pipet  10  c.c.  of  urine  into  an  ordi- 
nary conical  centrifuge  tube,  which  has  been  previously  weighed ; 
add  i  c.c.  of  5  per  cent,  acetic  acid,  and  let  stand  for  fifteen  min- 
utes in  a  beaker  of  boiling  water.  At  the  end  of  this  time  remove 
the  tube  from  the  water-bath  and  centrifuge  for  a  few  minutes. 
Pour  off  the  supernatant  liquid,  stir  up  the  precipitate  in  the  tube 
with  about  10  c.c.  of  boiling  .5  per  cent,  acetic  acid,  and  again 
centrifuge.  Remove  the  supernatant  liquid  from  the  precipitate 

173 


in  the  tube  and  wash  once  more,  this  time  with  50  per  cent,  alco- 
hol. After  centrifuging  and  pouring  off  the  supernatant  alcohol, 
place  the  tube  for  two  hours  in  an  air  bath  at  100-110°,  then  cool 
in  a  desiccator,  and  weigh. 

McCrudden's  Method  for  the  Determination  of  Calcium  and 
Magnesium  in  Urine  (/.  Blol.  Chem.,  7,  82  and  10,  187). — If  the 
urine  is  alkaline,  make  it  neutral  or  slightly  acid  to  litmus.  Fil- 
ter. Transfer  200  c.c.  of  the  filtered  urine  to  a  small  flask.  Make 
just  alkaline  with  concentrated  ammonium  hydrate  and  then  just 
acid  with  (concentrated)  hydrochloric  acid.  The  cloud  of  phos- 
phates forming  in  alkaline  urine  may  be  used  as  a  guide  in  the 
process  of  acidifying  the  urine.  Cool  for  a  few  minutes  in  run- 
ning water.  Add  10  drops  of  concentrated  hydrochloric  acid  and 
10  c.c.  of  2.5  per  cent,  oxalic  acid.  Now  add  8  c.c.  of  20  per 
cent,  sodic  acetate  solution,  stopper,  and  shake  vigorously  and 
continuously  for  about  ten  minutes.  Filter  on  a  small  ash  free 
filter  paper  and  wash  free  from  chlorids  with  .5  per  cent,  ammo- 
nium oxalate  solution.  Transfer  the  filter  and  precipitate  to  a 
•weighed  platinum  crucible,  dry  over  a  small  flame,  and  then  heat 
in  the  blast  lamp  to  constant  weight,  thus  transforming  the  cal- 
cium oxalate  to  calcium  oxid.  Cool  in  a  desiccator  and  weigh. 

In  the  combined  filtrate  and  washwater  the  magnesium  is  de- 
termined as  follows:  Transfer  the  filtrate  to  a  large  porcelain 
dish,  add  20  c.c.  concentrated  nitric  acid,  and  boil  down  almost  to 
dryness.  When  the  residue  is  nearly  dry  and  no  more  nitrous 
fumes  are  given  off,  add  10  c.c.  concentrated  hydrochloric  acid 
and  again  boil  down  nearly  to  dryness.  Dilute  with  water  to  a 
volume  of  almost  80  c.c.,  and  with  constant  stirring  add  ammonia, 
drop  by  drop,  until  the  mixture  is  alkaline  to  litmus  paper.  Then 
add  25  c.c.  dilute  ammonia  (sp.  gr.  .96)  slowly  and  with  stirring, 
and  set  aside  over  night  in  a  cool  place.  Filter  on  a  small  filter 
paper,  and  wash  the  precipitate  with  a  dilute  solution  of  alcohol 
and  ammonia  (i  volume  of  alcohol  and  i  volume  dilute  ammonia 
mixed  with  3  volumes  of  water).  Wash  until  the  filtrate  is  free 
from  chlorids.  Dry.  the  filter  and  ignite  in  a  weighted  platinum 
crucible.  Cool  and  weigh.  The  residue  is  Mg2P2O7. 

Method  for  tfie  Determination  of  Sodium  and  Potassium  in  Tlrine. 

— Transfer  50  c.c.  of  urine  to  a  platinum  dish  (capacity  about 
250  c.c.),  evaporate  to  dryness,  and  then  heat  the  residue,  at  first 

173 


very  cautiously,  over  a  radial  burner.  Continue  the  heating  at  a 
barely  perceptible  dull  red  heat  for  one  hour.  Cool.  Moisten  the 
residue  with  se-err:  distilled  water,  evaporate  to  dryness,  and  heat 
as  before  for  another  hour.  To  the  residue,  which  now  should 
contain  very  little  carbon,  add  5e-Tc.c.  water  and  5-6  drops  con- 
centrated hydrochloric  acid.  The  mineral  constituents  are  thus 
brought  into  solution.  Add  an  excess  of— saturated  barium  hy- 
droxid  sokttien-  (i.e.,  to  a  distinctly  alkaline  reaction),  heat  to 
boiling,  and  filter  on  a  Gooch  crucible.  Wash  with  hot  water. 
The  filtrate  should  now  be  substantially  free  from  calcium,  mag- 
nesium, phosphoric  acid,  and  sulphuric  acid,  but  does  contain 
barium  in  addition  to  the  sodium  and  potassium.  Precipitate  the 
barium  by  passing  wasked^earbonic-^cKr-through  the  solution. 
Filter  on  another  Gooch  crucible  and  wash  with  a  little  cold 
water.  Render  the  filtrate  slightly  acid  to  methyl  orange  (one 
drop),  and  evaporate  to  dryness  in  a  previously  weighed  plat- 
inum dish.  Heat  the  residue  very  gradually  and  carefully  to  a 
dull  red  heat  for  10  minutes.  Cool  in  a  desiccator  and  weigh. 
The  increase  in  weight  gives  the  sodium  and  potassium  as  chlo- 
rids. 

Dissolve  the  residue  in  a  very  small  quantity  of  water  and 
add  a  few  drops  dilute  hydrochloric  acid.  Then  add  10  per  cent. 
chlorplatinic  acid  solution  (4-5  times  as  much  H2PtCl6  as  the 
combined  weight  of  the  chlorids  present),  and  evaporate  at  me- 
dium temperature,  about  75°  C,  until  the  residue  looks  dry.  Now 
add  95  per  cent,  alcohol,  filter  on  a  weighed  Gooch  crucible,  and 
wash  several  times  with  95  per  cent,  alcohol.  Dry  at  no0  and 
weigh.  The  potassium  chlorplatinate  thus  obtained  multiplied  by 
the  factor  .3056  gives  the  corresponding  weight  of  potassium 
chlorid.  The  sodium  chlorid  is  then  obtained  by  subtracting  the 
weight  of  the  potassium  chlorid  from  the  weight  of  the  combined 
chlorids. 

In  connection  with  this  determination  there  are  two  fruitful 
sources  of  error :  contamination  of  the  chlorplatinate  precipitate  with 
ammonia  (which,  as  ammonium  chlorplatinate,  gives  too  high  results 
for  potassium),  and  overheating  during  the  ashing  (which  causes 
volatilization  of  the  sodium  chlorid).  Loss  of  chlorids  through  over- 
heating may  be  avoided  by  placing  the  platinum  dish  containing  the 
dried  urine  on  fragments  of  clay  plate,  or  pieces  of  a  broken  evapo- 
rating dish,  contained  in  a  shallow  iron  dish  (about  20  cm.  in  diam- 
eter) which  is  heated  by  means  of  a  large  size  radial  burner. 

177 


BLOOD 

Preparation  of  Protein-Free  Blood  Filtrates. —  (/.  Biol  Chem., 
38,  81,  1919).  The  blood  filtrate,  the  preparation  of  which  is 
described  below,  is  suitable  for  the  determination  of  non-protein 
nitrogen,  urea,  uric  acid,  creatinin,  creatin  and  sugar. 

The  blood  should  be  collected  over  finely  powdered  potassium 
oxalate,  about  20  mg.  for  10  c.c.  of  blood.  It  is  important  not 
to  use  unnecessarily  large  amounts  of  oxalate  because  the  excess 
makes  the  complete  coagulation  of  the  proteins  more  difficult 
and  also  interferes  more  or  less  with  the  uric  acid  precipitation. 

Reagents  required  for  the  precipitation  of  the  proteins: 

1.  A  10  per  cent,  solution  of  sodium  tungstate.    Some  sodium 
tungstates,  though  labeled  c.p.,  are  not  serviceable  for  this  work. 
They  usually   contain  too   much   sodium  carbonate.     The  c.p. 
sodium  tungstate  made  by  the  Primos  Chemical  Company,  Pri- 
mos,  Pa.,  is  satisfactory. 

2.  A   two-thirds   normal   sulphuric  acid   solution,   35   g.   of 
concentrated  c.p.  sulphuric  acid  diluted  to  a  volume  of  I  liter, 
will  usually  be  found  to  be  correct;  but  it  is  advisable,  indeed 
necessary,  to  check  it  up  by  titration.     The  two-thirds  normal 
acid  is  intended  to  be  equivalent  to  the  sodium  content  of  the 
tungstate  so  that  when  equal  volumes  are  mixed  substantially 
the  whole  of  the  tungstic  acid  is  set  free  without  the  presence 
of  an  excess  of  sulphuric  acid.     The  tungstic  acid  set  free  is 
nearly  quantitatively  taken  up  by  the  proteins  and  the  blood  fil- 
trates obtained   are  therefore  only  slightly  acid  to  congo  red 
paper. 

Transfer  a  measured  quantity  (5  to  15  c.c.)  of  oxalated  blood 
to  a  flask  having  a  capacity  of  fifteen  to  twenty  times  that  of  the 
volume  taken.  Lake  the  blood  with  seven  volumes  of  water. 
Add  one  volume  of  ip  per  cent,  solution  of  sodium  tungstate 
(Na2WO4,2H2O)  and  mix.  Add  from  a  graduated  pipet  or 
buret,  slowly  and  with  shaking,  one  volume  of  two-thirds 
normal  sulphuric  acid.  Close  the  mouth  of  the  flask  with  a 
rubber  stopper  and  shake.  If  the  conditions  are  right,  hardly 
a  single  air  bubble  will  form  as  a  result  of  the  shaking.  Let 
stand  for  5  minutes ;  the  color  of  the  coagulum  gradually  changes 
from  bright  red  to  dark  brown.  If  this  change  in  color  does 
not  occur,  the  coagulation  is  incomplete,  usually  because  too 
much  oxalate  is  present.  In  such  an  emergency  the  sample  may 

179 


if 


be  saved  by  adding  10  per  cent,  sulphuric  acid,  one  drop  at  a 
time  shaking  vigorously  after  each  drop,  and  continuing  until 
there  is  practically  no  foaming  and  until  the  dark  brown  color 
has  set  in. 

Pour  the  mixture  on  a  filter  large  enough  to  hold  it  all.  This 
filtration  should  be  begun  by  adding  only  a  few  c.c.  of  the  mix- 
ture down  the  double  portion  of  the  filter  paper  and  withholding 
the  remainder  until  the  whole  filter  has  been  wet.  Then  the 
whole  of  the  mixture  is  poured  on  the  funnel  and  covered  with  a 
watch  glass.  If  the  filtration  is  made  as  described  the  very  first 
portion  of  the  filtrate  should  be  clear  as  water  and  no  re-filtering 
is  necessary. 

It  will  be  noted  that  the  precipitation  is  not  made  in  volu- 
metric flasks.  By  the  process  described  6  or  7  or  n  or  12  c.c.  of 
blood  can  be  used,  whereas  with  volumetric  flasks  one  is  com- 
pelled to  use  5,  10  or  20  c.c.,  because  flasks  suitable  for  other 
volumes  are  not  available.  Special  graduated  "blood  pipets," 
made  by  the  Emil  Greiner  Co.,  New  York,  are  very  useful  for 
the  measurement  of  the  blood,  the  tungstate  and  the  acid. 

The  protein  blood  filtrates  are  not  acid  enough  to  prevent  bac- 
terial decomposition.  If  the  filtrates  are  to  be  kept  for  any 
length  of  time,  more  than  two  days,  some  preservative,  a  few 
drops  of  toluene  or  xylene  should  be  added. 

Determination  of  Non-protein  Nitrogen. — For  the  digestion  of 
5  c.c.  of  blood  filtrate  it  is  not  necessary  to  use  more  than  one- 
half  c.c.  of  the  phosphoric-sulphuric  acid  mixture  described  on 
p.  27.  Dilute  50  c.c.  of  the  acid  mixture  with  50  c.c.  of  water 
and  keep  well  protected  to  prevent  the  absorption  of  ammonia. 
Use  i  c.c.  for  each  digestion. 

The  digestion  is  most  conveniently  made  in  ignition  test  tubes 
(Pyrex,  200  mm.  x  25  mm.)  which  have  been  graduated  at  35 
c.c.  and  at  50  c.c.  Such  test  tubes  can  be  obtained  from  the 
Emil  Greiner  Co.,  New  York. 

Transfer  5  c.c.  of  the  blood  filtrate  to  such  a  test  tube.  The 
test  tube  should  either  be  dry  or  rinsed  with  alcohol  to  reduce 
the  danger  of  bumping.  Add  i  c.c.  of  the  diluted  acid  mixture 
and  a  quartz  pebble.  Boil  vigorously  over  a  micro  burner  until 
the  characteristic  dense  fumes  begin  to  fill  the  tube.  This  will 
happen  in  from  3  to  7  mirrtkes,  depending  on  the  size  of  the  flame. 
When  the  test  tube  is  nearly  full  of  fumes  reduce  the  flame 

181 


rv 


sharply  so  that  the  speed  of  the  boiling  is  reduced  almc«$  to 
the  vanishing  point.  Cover  the  mouth  of  the  test  tube  with  a 
watch  glass.  Continue  the  gentle  heating  for  2  minutes,  counting 
from  the  time  the  test  tube  became  filled  with  fumes.  If  the 
oxidations  are  not  visibly  finished  at  the  end  of  two  minutes  the 
heating  must  be  continued  until  the  solution  is  nearly  colorless. 
Usually  the  solution  becomes  colorless  at  the  end  of  20  to  40 
seconds.  At  the  end  of  2  minutes  remove  the  flame  and  allow 
the  digestion  mixture  to  cool  for  70  to  90  seconds.  Then  add 
15  to  25  c.c.  of  water.  Cool  further  approximately  to  room 
temperature  and  then  fill  to  the  35  c.c.  mark  with  water.  Add 
15  c.c.  of  Nessler's  solution  (p.  203).  Insert  a  clean  rubber 
stopper  and  mix.  If  the  solution  is  turbid,  centrifuge  a  portion 
before  making  the  color  comparison  with  the  standard. 

The  standard  most  commonly  required  is  0.3  mg.  of  N.  Add 
3  c.c.  of  the  standard  ammonium  sulphate  solution  (containing 
i  mg.  of  N  per  10  c.c.)  to  a  100  c.c.  volumetric  flask.  Add  to 
it  2  c.c.  of  the  phosphoric  sulphuric  acid  mixture,  to  balance  the 
acid  in  the  test  tube ;  dilute  to  about  60  c.c.  and  add  30  c.c.  of 
Nessler's  solution.  The  unknown  and  the  standard*  should  be 
Nesslerized  simultaneous^ 

Calculation. — If  the  standard  is  set  at  20  mm.  for  the  color 
comparison,  20  divided  by  the  reading  and  multiplied  by  0.3 
gives  the  non-protein  nitrogen  in  I  c.c.  of  blood,  because  0.5 
c.c.  (the  amount  of  blood  represented  in  5  c.c.  of  the  blood  fil- 
trate) Nesslerized  at  a  volume  of  50  c.c.  is  equivalent  to  i  c.c. 
Nesslerized  at  a  volume  of  100  c.c. 

The  non-protein  nitrogen  per  100  c.c.  of  blood  is  therefore  20 
divided  by  the  reading  and  multiplied  by  30  (0.3  times  100). 

If  the  standard  containing  0.5  mg.  N  is  used  the  calculation  be- 
comes 20,  divided  by  R,  times  50. 

DETERMINATION  OF  UREA.— Transfer  5  c.c.  of  the 
tungstic  acid  blood  filtrate  to  a  Pyrex  ignition  tube  (200  x  25 
mm.).  -This  test  tube  must  be  rinsed  with  nitric  acid  and  then 
with  water  if  it  has  contained  Nessler  Solution.  Add  2  drops 
of  buffer  mixture  (p.  107)  and  then  introduce  I  c.c.  of  urease 
solution  (p.  107).  Immerse  the  test  tube  in  warm  water,  40  to 
55°  C,  and  leave  it  there  for  5  minutes,  or  let  stand  at  room  tem- 
perature for  15  minutes. 

183 


AT  BEGINNING;  B,  TOWARD  END  OF  DISTILLATION 

185 


\  ) 


The  ammonia  formed  from  the  urea  is  most  conveniently  ob- 
tained by  distillation,  without  a  condenser,  and  using  a  test 
tube  graduated  at  25  c.c.  and  containing  2  c.c.  of  0.05  X  hydro- 
chloric acid  as  the  receiver.  The  illustration  shows  a  compact 
and  convenient  arrangement  for  this  distillation. 

Add  to  the  urease  blood  filtrate  a  dry  pebble,  a  drop  or  two  of 
paraffin  oil  and  2  c.c.  of  saturated  borax  solution.  Insert'firmly 
the  rubber  stopper  carrying  the  delivery  tube  and  receiver  and 
then  boil  at  a  moderately  fast,  uniform  rate  for  4  minutes.  The 
size  of  the  flame  should  never  be  cut  down  during  the  distilla- 
tion, nor  should  the  boiling  be  so  brisk  that  the  emission  of  steam 
from  the  receiver  begins  before  the  end  of  3  minutes.  At  the 
end  of  4  minutes  slip  off  the  receiver  from  the  rubber  stopper 
and  let  it  rest  in  a  slanting  position  while  the  distillation  is  con- 
tinued for  i  more  minute.  Rinse  the  lower  end  of  the  delivery 
tube  with  a  little  water  and  cool  the  distillate  with  running 
water  and  dilute  to  about  20  c.c.  Transfer  0.3  mg.  N  (3  c.c.  of 
the  standard  ammonium  sulphate  solution)  to  a  100  c.c.  volu- 
metric flask  and  dilute  to  about  75  c.c.  Nesslerize,  using  10  c.c.  of 
Xessler's  Solution  for  the  Standard,  and  2.5  c.c,  for  the  unknown 
in  the  test  tube.  Dilute  both  to  volume  and  make  the  color  com- 
parison. 

Calculation. — Divide  20  (the  height  of  the  standard  in  mm.) 
by  the  colorimetric  reading  and  multiply  by  15.  This  gives  the 
urea  nitrogen  in  mgs.  per  100  c.c.  of  blood.  In  explanation  of 
this  calculation  it  is  to  be  noted  that  the  unknown  representing  0.5 
c.c.  of  blood,  is  Xesslerized  at  25  c.c.,  whereas  in  the  case  of 
the  non-protein  nitrogen  it  is  Xesslerized  at  a  volume  of  50 
c.c.  The  same  colorimetric  reading  therefore  represents  only 
one-half  as  much  nitrogen  in  the  urea  determination  as  in  the 
non-protein  nitrogen  determination. 


Urea  Determination  by  Means  of  the  Autoclave. — When  a 
large  number  of  urea  determinations  are  to  be  made  or  when 
creatin  determinations  are  also  made,  it  is  sometimes  con- 
venient to  decompose  the  urea  of  the  blood  filtrate  by  heating 
under  pressure.  To  5  c.c.  of  the  blood  filtrate  in  a  large  test- 
tube  add  I  c.c.  of  normal  hydrochloric  acid,  cover  with  tin  foil 
and  heat  to  150°  for  10  minutes.  Distil  off  the  ammonia  ex- 

187 


actly  as  in  the  preceding  process,  except  that  2  c.c.  of  10  per 
cent,  sodium  carbonate  must  be  substituted  for  the  borax,  because 
of  the  added  hydrochloric  acid. 

Aeration  Process  in  Urea  Determination. — The  removal  of  the 
ammonia  formed  from  the  blood  urea  by  urease,  or  by  heating 
under  pressure,  can,  of  course,  be  driven  into  the  receiver  by  an 
air  current  plus  an  alkali,  instead  of  by  the  distillation  process 
described  above.  The  aeration  process  gives  perfectly  reliable 
results,  if  a  good  air  current  is  available. 

To  the  decomposed  blood  filtrate  in  a  large  test  tube  add  a 
little  paraffin  oil  and  i  or  2  c.c.  of  10  per  cent,  sodium  hydroxid. 
Connect  with  a  smaller  test  tube,  marked  at  25  c.c.,  and  contain- 
ing 2  c.c.  of  0.5  N  hydrochloric  acid.  The  connection  is  made 
as  in  the  macro  aeration  process  (see  p.  91).  Pass  the  air  cur- 
rent through  rather  slowly  for  I  minute  and  then  nearly  as  fast 
as  the  apparatus  can  stand  for  10  to  15  minutes.  Rinse  the  con- 
necting tube ;  dilute  the  contents  of  the  receiver  to  20  c.c.,  add 
2.5  c.c.  of  Nessler  Solution,  dilute  to  the  25  c.c.  mark,  and  make 
the  color  comparison  in  the  usual  manner. 

DETERMINATION  OF  PREFORMED  CREATININ.— 
Transfer  25  (or  50)  c.c.  of  a  saturated  solution  of  purified  picric 
acid  to  a  small,  clean  flask,  add  5  (or  10)  c.c.  of  10  per  cent,  so- 
dium hydroxid,  and  mix.  Transfer  10  c.c.  of  blood  filtrate  to  a 
small  flask  or  to  a  test  tube,  transfer  5  c.c.  of  the  standard  crea- 
tinin  solution  described  below  to  another  flask,  and  dilute  the 
standard  to  20  c.c.  Then  add  5  c.c.  of  the  freshly  prepared 
alkaline  picrate  solution  to  the  blood  filtrate,  and  10  c.c.  to  the 
diluted  creatinin  solution.  Let  stand  for  8  to  10  minutes  and 
make  the  color  comparison  in  the  usual  manner,  never  omitting 
first  to  ascertain  that  the  two  fields  of  the  colorimeter  are  equal 
when  both  cups  contain  the  standard  creatinin  picrate  solution. 
The  color  comparison  should  be  completed  within  15  minutes 
from  the  time  the  alkaline  picrate  was  added;  it  is  therefore 
never  advisable  to  work  with  more  than  three  to  five  blood  fil- 
trates at  a  time 

When  the  amount  of  blood  filtrate  available  for  the  creatinin 
determination  is  too  small  to  permit  repetition,  it  is  of  course 
advantageous  or  necessary  to  start  with  more  than  one  standard. 
If  a  high  creatinin  should  be  encountered  unexpectedly  without 

189 


several  standards  ready,  the  determination  can  be  saved  by  dilut- 
ing the  unknown  with  an  appropriate  amount  of  the  alkaline 
picrate  solution — using  for  such  dilution  a  picrate  solution  first 
diluted  with  two  volumes  of  water — so  as  to  preserve  equality  be- 
tween the  standard  and  the  unknown  in  relation  to  the  concen- 
tration of  picric  acid  and  sodium  hydroxid. 

One  standard  creatinin  solution,  suitable  both  for  creatinin 
and  for  creatinin  determinations  in  blood,  can  be  made  as  fol- 
lows: Transfer  to  a  liter  flask  6  c.c.  of  the  standard  creatinin 
solution  used  for  urine  analysis  (which  contains  6  mg.  of  crea- 
tinin) ;  add  10  c.c.  of  normal  hydrochloric  acid,  dilute  to  the 
mark  with  water,  and  mix.  Transfer  to  a  bottle  and  add  four 
or  five  drops  of  toluene  or  xylene.  5  c.c.  of  this  solution  con- 
tain 0.03  mg.  of  creatinin,  and  this  amount  plus  15  c.c.  of  water 
represents  the  standard  needed  for  the  vast  majority  of  human 
bloods,  for  it  covers  the  range  of  i  to  2  mg.  per  100  c.c.  In  the 
case  of  unusual  bloods  representing  retention  of  creatinin,  take 
10  c.c.  of  the  standard  plus  10  c.c.  of  water,  which  covers  the 
range  of  2  to  4  mg.  of  creatinin  per  100  c.c.  of  blood;  or  15 
c.c.  of  the  standard  plus  5  c.c.  of  water  by  which  4  to  6  mg. 
can  be  estimated.  By  taking  the  full  20  c.c.  volume  from  the 
standard  solution  at  least  8  mg.  can  be  estimated ;  but  when  work- 
ing with  such  blood  it  is  well  to  consider  whether  it  may  not  be 
more  advantageous  to  substitute  5  c.c.  of  blood  filtrate  plus  5 
c.c.  of  water  for  the  usual  10  c.c.  of  blood  filtrate. 

Calculation. — The  reading  of  the  standard  in  mm.  (usually 
20)  multiplied  by  1.5,  3,  4.5,  or  6  (according  to  how  much  of  the 
standard  solution  was  taken),  and  divided  by  the  reading  of  the 
unknown,  in  m.m.,  gives  the  amount  of  creatinin,  in  mg.  per 
100  c.c.  of  blood.  In  connection  with  this  calculation  It  is  to  be 
noted  that  the  standard  is  made  up  to  twice  the  volume  of  the  un- 
known, so  that  each  5  c.c.  of  the  standard  creatinin  solution, 
while  containing  0.03  mg.,  corresponds  to  0.015  mg.  in  the  blood 
filtrate. 

DETERMINATION  OF  CREATIN  PLUS  CREATININ. 
— Transfer  5  c.c.  of  blood  filtrate  to  a  test  tube  graduated  at  25 
c.c.  These  test  tubes  are  also  used  for  urea  and  for  sugar  de- 
terminations. Add  i  c.c.  of  normal  hydrochloric  acid.  Cover  the 
mouth  of  the  test  tube  with  tin- foil  and  heat  in  the  autoclave  to 
130°  C.  for  20  minutes  or,  as  for  the  urea  hydrolysis,  to  155°  C. 

191 


for  10  minutes.  Cool.  Add  5  c.c.  of  the  alkaline  picrate  solution 
and  let  stand  for  8  to  10  minutes,  then  dilute  to  25  c.c.  The 
standard  solution  required  is  10  c.c.  of  creatinin  solution  in  a  50 
c.c.  volumetric  flask.  Add  2  c.c.  of  normal  acid  and  10  c.c.  of  the 
alkaline  picrate  solution  and  after  10  minutes  standing  dilute  to 
50  c.c.  The  preparation  of  the  standard  must  of  course  have 
been  made  first  so  that  it  is  ready  for  use  when  the  unknown  is 
ready  for  the  color  comparison.  The  height  of  the  standard, 
usually  20  mm.,  divided  by  the  reading  of  the  unknown  and  mul- 
tiplied by  6  gives  the  "total  creatinin"  in  mg.  100  c.c.  blood. 

In  the  case  of  uremic  bloods  containing  large  amounts  of  crea- 
tinin i,  2,  or  3  c.c.  of  blood  filtrate,  plus  water  enough  to  make 
approximately  5  c.c.,  are  substitutes  for  5  c.c.  of  the  undiluted 
filtrate. 

The  normal  value  for  "total  creatinin"  given  by  this  method 
is  about  6  mg.  per  i  jo  c.c.  of  blood. 

Determination  of  Uric  Acid  in  Blood. — Solutions  Required  for 
Uric  Acid  Determinations. 

1.  The  standard  uric  acid  sulphite  solution  already  described 

(P-  "5-) 

2.  A  10  per  cent,  sodium  sulphite  solution. 

3.  A  5  per  cent,  sodium  cyanid  solution,  to  be  added  from  a 
buret. 

4.  A  10  per  cent,  solution  of  sodium  chlorid  in  o.i  normal 
hydrochloric  acid. 

5.  The   uric  acid   reagent   prepared  according  to  Folin  and 
Denis  (see  p.  207).    A  still  stronger  reagent  is  obtained  by  heat- 
ing the   sodium  tungstate    (100  gm.)    and  the  phosphoric  acid 
(80  c.c.)  plus  water  (700  c.c.)  for  24  hours,  instead  of  2  hours; 
but  the  advantage  gained,  about  20  per  cent.,  is  not  needed.    Di- 
lute the  solution  to  i   liter. 

6.  A  solution  of  5  per  cent,  silver  lactate  in  5  per  cent,  lactic 
acid. 

To  10  c.c.  of  blood  filtrate  in  each  of  two  centrifuge  tubes  add 
2  c.c.  of  a  5  per  cent,  solution  of  silver  lactate  in  5  per  cent,  lactic 
acid,  and  stir  with  a  very  fine  glass  rod.  Centrifuge;  add  a 
drop  of  silver  lactate  to  the  supernatant  solution,  which  should  be 
almost  perfectly  clear  and  should  not  become  turbid  when  the' 
last  drop  of  silver  solution  is  added.  Remove  the  supernatant 

193 


liquid  by  decantation  as  completely  as  possible.  Add  to  each 
tube  i  c.c.  of  a  solution  of  10  per  cent,  sodium  chlorid  in  o.i 
normal  hydrochloric  acid  and  stir  thoroughly  with  the  glass  rod. 
Then  add  5  to  6  c.c.  of  water,  stir  again,  and  centrifuge  once 
more.  By  this  chlorid  treatment  the  uric  acid  is  set  free  from 
the  precipitate.  Transfer  the  two  supernatant  liquids  by  decanta- 
tion to  a  25  c.c.  volumetric  flask.  Add  i  c.c.  of  a  10  per  cent. 
solution  of  sodium  sulphite,  0.5  c.c.  of  a  5  per  cent,  solution  of  so- 
dium cyanid,  and  3  c.c.  of  a  20  per  cent,  solution  of  sodium  car- 
bonate. Prepare  simultaneously  two  standard  uric  acid  solutions 
as  follows: 

Transfer  to  one  50  c.c.  volumetric  flask  i  c.c.  and  to  another  50 
c.c.  flask  2  c.c.  of  the  standard  uric  acid  sulphite  solution  described 
above.  To  the  first  flask  add  also  i  c.c.  of  10  per  cent,  sodium 
sulphite  solution.  Then  add  to  each  flask  4  c.c.  of  the  acidified  so- 
dium chlorid  solution,  i  c.c.  of  the  sodium  cyanid  solution,  and 
6  c.c.  of  the  sodium  carbonate  solution.  Dilute  with  water  to 
about  45  c.c.  When  the  two  standard  solutions  and  the  unknown 
have  been  prepared  as  described  they  are  ready  for  the  addition 
of  the  uric  acid  reagent.  Add  0.5  c.c.  of  this  reagent  to  the 
unknown  and  i  c.c.  to  each  of  the  standards,  and  mix.  Let  stand 
for  10  minutes,  fill  to  the  mark  with  water,  mix,  and  make  the 
color  comparison. 

Calculation. — In  connection  with  the  calculation  it  is  to  be 
noted  (a)  that  the  blood  filtrate  taken  corresponds  to  2  c.c.  of 
blood,  (b)  that  the  standard  is  diluted  to  twice  the  volume  of  the 
unknown,  and  (c)  that  the  standard  used  contains  o.i  or  0.2  mg. 
of  uric  acid.  The  blood  filtrate  from  blood  containing  2.5  mg.  of 
uric  acid  will  be  just  equal  in  color  to  the  weaker  standard.  20 
times  2.5  divided  by  the  reading  of  the  unknown  gives,  therefore, 
the  uric  acid  content  of  the  blood  when  the  weaker  standard  is 
set  at  20  mm. 

The  two  standards  recommended  were  adopted  on  the  basis  of 
the  experience  gained  from  the  analysis  of  more  than  150  dif- 
ferent samples  of  human  blood.  The  uric  acid  may  sink 
to  as  low  as  i  mg.  of  uric  acid  per  100  c.c.  of  blood.  It  seems 
hardly  worth  while  to  prepare  a  third  and  weaker  standard  regu- 
larly in  order  to  provide  for  such  low  acid  values.  A  stand- 
ard corresponding  to  the  color  obtained  from  1.25  mg.  of  uric 
acid  per  100  c.c.  of  blood  can  be  prepared  within  a  couple  of 
minutes  as  follows :  Transfer  i  c.c.  of  10  per  cent,  sulphite  solu- 

195 


tion,  3  c.c.  of  20  per  cent,  sodium  carbonate,  2  c.c.  of  the  acidi- 
fied sodium  chlorid,  0.5  c.c.  of  the  sodium  cyanid  solution,  and 
25  c.c.  of  the  weaker  one  of  the  two  "regular  standard  solutions 
already  on  hand.  Dilute  to  50  c.c.  and  mix.  Or,  simply  add  5 
c.c.  of  20  per  cent,  sodium  carbonate  to  25  c.c.  of  the  regular 
weaker  standard,  and  dilute  to  50  c.c. 

If  a  low  uric  acid  value  is  expected,  an  alternate  procedure  is 
to  dilute  the  unknown  to  a  final  volume  of  10  c.c.  with  corre- 
sponding reduction  in  the  amount  of  the  reagents  used. 

Special  attention  should  perhaps  be  called  to  one  small  yet  es- 
sential variation  in  the  process  for  developing  the  blue  uric  acid 
color,  a  variation  made  necessary  by  the  use  of  sodium  sulphite. 
The  uric  acid  reagent  must  .invariably  be  added  after,  and  not 
before,  the  addition  of  the  sodium  carbonate,  because  in  acid 
solution  the  sulphite  will  itself  give  a  blue  color  with  phospho- 
tungstic  acid. 

New  Method  for  Determination  of  Sugar  in  Blood, — Solutions 
needed: 

1.  Saturated  sodium  carbonate  solution. 

2.  Standard  Sugar  Solution. — Dissolve  I  gm.  of  pure  anhy- 
drous dextrose  in  water  and  dilute  to   a  volume  of  100  c.c.    Mix, 
add  a  few  drops  of  xylene  or  toluene,  and  bottle.     If  pure  dex- 
trose is  not  available,  a  standard  solution  of  invert  sugar  made 
from  cane  sugar  is  equally  useful.     Transfer  exactly  I  gm.  of 
cane  sugar  to  a  100  c.c.  volumetric  flask ;  add  20  c.c.  of  normal 
hydrochloric  acid  and  let  the  mixture  stand  over  night  at  room 
temperature  (or  rotate  the  flask  and  contents  continuously  for 
10  minutes  in  a  water  bath  kept  at  70°  C).    Add  1.68  gm.  of 
sodium  bicarbonate  and  about  0.2  gm.  of  sodium  acetate,  to 
neutralize  the  hydrochloric  acid.    Shake  a  few  minutes  to  remove 
most  of  the  carbonic  acid  and  fill  to  the  100  c.c.  mark  with  water. 
Then  add  5  c.c.  more  of  water  (i  gm.  of  cane  sugar  yields  1.05 
gm.  of  invert  sugar)  and  mix.    Transfer  to  a  bottle ;  add  a  few 
drops  of  xylene  or  toluene,  shake  well,  and  stopper  tightly.    The 
stock  solution  made  in  either  way  keeps  indefinitely.     Dilute  <f 
c.c.   to    500   c.c.,   giving  a   solution    10  c.c.   of   which   contain 
i  mg.  of  dextrose  or  invert  sugar.    Add  some  xylene.    Use  2  c.c. 
for  each  determination. 

3.  Alkaline  Copper  Solution.— Dissolve  40  gm.  of  anhydrous 

197 


m 


sodium  carbonate  in  about  400  c.c.  of  water  and  transfer  to  a 
liter  flask.  Add  7.5  gm.  of  tartaric  acid  and  when  the  latter  has 
dissolved  add  4.5  gm.  of  crystallized  copper  sulphate;  mix,  and 
make  up  to  a  volume  of  i  liter.  If  the  carbonate  used  is  impure, 
a  sediment  may  be  formed  in  the  course  of  a  week  or  so.  If  this 
happens,  decant  the  clear  solution  into  another  bottle. 

4.  Phosphotungstic-phosphomolybdic  Acid. — Transfer  to  a 
large  flask  25  gm.  of  molybdenum  trioxid  (MoO3)  or  34  gm.  of 
ammonium  molybdate  (NH4)2(MoO4)  ;  add  140  c.c.  of  10  per 
cent,  sodium  hydroxid  and  about  150  c.c.  of  water.  Boil  for 
20  minutes  to  drive  off  the  ammonia  (molybdic  acid  sometimes 
contains  large  amounts  of  ammonia  as  impurity).  Add  to  the 
solution  100  gm.  of  sodium  tungstate,  50  c.c.  of  85  per  cent, 
phosphoric  acid,  and  100  c.c.  of  concentrated  hydrochloric  acid. 
Dilute  to  a  volume  of  700  to  800  c.c. ;  close  the  mouth  of  the 
flask  with  a  funnel  and  watch-glass.  Boil  gently  for  not  less 
than  4  hours,  adding  hot  water  from  time  to  time  to  replace  that 
lost  during  the  boiling.  Cool  and  dilute  to  i  liter.  This  solution 
is  identical  with  the  phenol  reagent  of  Folin  and  Denis.  For 
use  in  connection  with  the  determination  of  blood  sugar  dilute 

1  volume  (100  c.c.)  of  the  reagent  with  one-half  volume  (50  c.c.) 
of  water  and  one-half  volume  (50  c.c.)  of  concentrated  hydro- 
chloric acid. 

The  determination  of  blood  sugar  is  carried  out  as  follows: 
Heat  a  beaker  of  water  to  vigorous  boiling.  Transfer  2»c.c.  of 
the  tungstic  acid  blood  filtrate  to  a  test-tube  (20  m.  X  206  mm.) 
graduated  at  25  c.c.  The  graduated  test-tubes  used  as  receivers 
when  distilling  off  the  ammonia  in  urea  determinations  (p.  185) 
are  suitable  for  this  work.  Transfer  2  c^c.  of  the  dilute  standard 
sugar  solution  to  another  similar  test-tube.  Add  to  each  tube 

2  c.c.  of  the  alkaline  copper  tartrate  solution.    Heat  in  the  boiling 
water  for  6  minutes.     Remove  the  test-tubes  and  add  at  once       , 
(without  cooling),  preferably   from  a  graduated  pipet,    i    c.c. 

of  the  strongly  acidified  and  diluted  phenol  reagent.  This 
should  be  done  as  nearly  simultaneously  as  possible ;  it  is  not 
advisable  to  use  one  standard  for  a  set  of  more  than  four  deter- 
minations. The  purpose  of  the  added  hydrochloric  acid  in  the 
reagent  is  to  dissolve  the  cuprous  oxide.  Mix,  cool,  and  add 
5  c.c.  of  saturated  sodium  carbonate  solution.  An  intense  blue 
color  is  gradually  developed  which  will  remain  unaltered  for 

199 


several  days.  Dilute  the  contents  of  both  test-tubes  to  the  25  c.c. 
mark,  and  after  at  least  5  minutes  make  the  color  comparison  in 
the  usual  manner. 

The  depth  of  the  standard  (in. mm.)  multiplied  by  100  and 
divided  by  the  reading  of  the  unknown  gives  the  sugar  content, 
in  mg.,  per  100  c.c.  of  blood. 


Sample   Analyses   of  Protein-Free   Blood    Filtrates   Obtained   by   Means   of 

Tungstic  Acid. 


No. 

Mg.  per  100  c.c.  blood  . 

Total  N. 

Urea  N. 

Uric  acid. 

Preformed 
creatinin. 

Total 
creatinin. 

Sugar. 

i 

26 

10 

1-3 

v   1.5 

6.0 

89 

2 

26 

13 

I.O 

•4 

5-3 

100 

3 

28 

12 

I.I 

.2 

6.7 

98 

4 

.28 

12 

2.2 

.0 

5-7 

83 

5 

29 

13 

3-3 

•5 

6.0 

86 

6 

29 

II 

2.6 

•4 

5-2 

95 

7 

29 

13 

1.6 

•4 

6.0 

85 

8 

30 

13 

2.4 

.6 

5-5 

82 

9 

30 

14 

4.1 

•  7 

5-3 

82 

10 

32 

15 

2.8 

.6 

5-4 

91 

ii 

32 

15 

3-4 

•4 

5-3 

97 

12 

32 

13 

2-4 

•7 

6.0 

104 

13 

33 

17 

2.0 

.3 

4.8 

'  83 

14 

33 

16 

2-5 

.6 

5-7 

105 

15 

33 

15 

I.I 

.6 

5-5 

95 

16 

34 

16 

0.8 

•  3 

6.1 

119 

17 

34 

16 

2.6 

•5 

5-9 

106 

18 

35 

17 

2.1 

.6 

'   6.0 

.89 

19 

20 

35 
35 

17 
18 

2.O 
2.0 

•4 
.  -7 

•  5-5 

'5-7 

11 

21 

35 

18 

2.9 

.6 

95 

22 

35 

17 

3-2 

•4 

lis 

94 

23 

35 

18 

2-5 

•  5 

6.0 

89 

24 

35 

19 

2.2 

•5 

5-3 

91 

25 

35 

22 

3-5 

•4 

5-7 

'87 

26 

35 

17 

2.3 

•  7 

6-7 

83 

27 

35 

18 

1.6 

•3 

6-5 

104 

28 

36 

17 

2.8 

•  5 

5-2 

IOO 

29 

37 

18 

2.1 

•5 

,5-5 

94 

30 

38 

18 

2.2 

•  7 

95 

31 

39 

18 

2.6 

.8 

6'-7 

103 

32 

39 

18 

2.9 

•  5 

6.0 

87 

33 

40 

18 

2.0 

.6 

6.0 

98 

34 

40 

20 

2.6 

•7 

5-6 

95 

35 

41 

19 

4.8 

•5 

5-9 

93 

36 

41 

19 

4-2 

2-5 

6.6 

109 

37 

43 

19 

2.2 

1-7 

6.3 

78 

38 

139 

106 

5-4 

12.5 

19.4 

99 

39 

147 

"5 

8.9 

II.  0 

20.5 

170 

t 

275 

237 

14-3 

13-6 

27.2 

157 

201 


The  Determination  of  Ammonia  in  Blood. — /.  BioL  Chem.,  n,- 
534.) — Reasonably  accurate  determinations  of  ammonia  in  blood  are 
obtained  with  great  difficulty  because  of  the  decomposition  of  certain 
nitrogenous  components  of  blood  even  at  room  temperatures,  and 
because  the  free  ammonia  actually  present  in  fresh  blood  amounts 
only  to  a  few  hundredths  of  a  milligram  per  100  c.c. 

Transfer  10  c.c.  of  blood  to  a  large  test  tube.  Add  2-3  c.c.  of 
a  solution  containing  10  per  cent,  sodic  carbonate  and  15  per  cent, 
potassium  oxalate.  Then  aspirate  the  liberated  ammonia  by 
means  of  a  rapid  air  current  into  a  test  tube  containing  i  c.c.  of 
water  and  5-6  drops  .1  N  hydrochloric  acid.  Time  20-30  min- 
utes. Nesslerize  this  solution  by  the  gradual  addition  of  not  over 
i  c.c.  diluted  Nessler's  solution  (dilution  1:5).  Transfer  the 
solution  to  a  10  c.c.  volumetric  flask,  and  dilute  to  volume  with 
"ammonia  free"  water. 

"Ammonia  free"  water  is  obtained  from  ordinary  distilled  water 
by  the  addition  of  a  little  bromin  water  and  a  few  drops  of  concen- 
trated sodium  hydroxid. 

The  colorimetric  valuation  of  the  solution  by  means  of  the 
Duboscq  colorimeter  cannot  be  accomplished  without  materially 
altering  the  instrument.  An  iris  diaphragm  should  be  attached 
to  one  sliding  platform  of  the  colorimeter,  so  as  to  regulate  the 
amount  of  light  passing  through  on  that  side.  The  hexagonal 
prism  should  be  removed  from  the  opposite  side.  With  these 
alterations,  the  Nesslerized  solution  in  a  100  mm.  polariscope 
tube  may  be  compared  with  .5  or  i  mg.  ammonia  (Nesslerized 
and  diluted  to  100  c.c.).  Place  the  standard  in  the  cup  on  the 
side  of  the  iris  diaphragm,  fill  a  100  mm.  polariscope  tube  with 
the  unknown,  and  insert  this  on  the  other  side.  Adjust  the 
standard  until  the  two  fields  are  equal. 

Nessler's  Reagent. — This  reagent  is  essentially  a  solution  of  the 
double  iodid  of  mercury  and  potassium  (HgI2,2KI)  containing 
sodic  or  potassic  hydrate.  A  stock  solution  of  the  double  iodid 
is  best  prepared  as  follows: 

Transfer  150  g.  of  potassium  iodid  and  no  g.  of  iodin  to  a 
500  c.c.  Florence  flask ;  add  100  c.c.  of  water  and  an  excess  of 
metallic  mercury,  140  g.  to  150  g.  Shake  the  flask  continuously 
and  vigorously  for  7  to  15  minutes  or  until  the  dissolved  iodin 

203 


has  nearly  all  disappeared.  The  solution  becomes  quite  hot. 
When  the  red  iodin  solution  has  begun  to  become  visibly  pale, 
though  still  red,  cool  in  running  water  and  continue  the  shaking 
until  the  reddish  color  of  the  iodin  has  been  replaced  by  the 
greenish  color  of  the  double  iodid.  This  whole  operation  usually 
does  not  take  more  than  15  minutes.  Now  separate  the  solution 
from  the  surplus  mercury  by  decantation  and  washing  with 
liberal  quantities  of  distilled  water.  Dilute  the  solution  and 
washings  to  a  volume  of  two  liters.  If  the  cooling  was  begun 
in  time  the  resulting  reagent  is  clear  enough  for  immediate  dilu- 
tion with  10  per  cent,  alkali  and  water,  and  the  finished  solution 
can  at  once  be  used  for  Nesslerizations. 

The  cost  of  the  chemicals  called  for  in  this  rather  interesting 
process  of  making  Nessler's  solution  is  less  than  when  starting 
with  mercuric  iodid  and  the  disagreeable  impurities  present  in 
many  samples  of  mercuric  iodid  are  avoided.  From  the  stock 
solution  of  mercuric  potassium  iodid,  made  as  described  above, 
prepare  the  final  Nessler  solution  as  follows : 

From  completely  saturated  caustic  soda  solution  containing 
about  55  g.  of  NaOH  per  100  c.c.  decant  the  clear  supernatant 
liquid  and  dilute  to  a  concentration  of  10  per  cent.  (It  is  worth 
while  to  determine  by  titration  that  a  10  per  cent,  solution  has 
been  obtained  with  an  error  of  not  over  5  per  cent.)  Introduce 
into  a  large  bottle  3,500  c.c.  of  10  per  cent,  sodic  hydrate  solu- 
tion, add  750  c.c.  of  the  double  iodid  solution,  and  750  c.c.  of 
distilled  water,  giving  5  liters  of  Nessler's  solution. 

In  the  absence  of  modifying  circumstances,  such  as  the  pres- 
ence of  much  acid  or  alkali,  this  reagent  should  be  added  in  the 
proportion  of  10  c.c.  per  100  c.c.  of  the  volume  to  which  the 
Nesslerized  solution  is  to  be  diluted.  As  a  general  rule  the 
volumetric  flask  (or  volumetric  test  tube)  should  be  at  least 
two-thirds  full  before  adding  the  Nessler  reagent.  If  attention 
is  not  given  to  this  detail  turbid  mixtures  are  obtained,  and  turbid 
solutions  must  never  be  used  for  color  comparisons. 

Preparation  of  Uric  Acid  and  Phenol  Reagent — Transfer  to  a 
flask  (capacity  about  1500  c.c.)  : 
750  c.c.  of  water, 
100  g.  of  sodium  tungstate, 
20  g.  of  phosphomolybdic  acid, 
50  c.c.  of  phosphoric  acid  (85  per  cent.  H3PO4), 
TOO  c.c.  of   concentrated   hydrochloric   acid. 
205 


Insert  a  funnel  in  the  flask  and  partly  close  the  opening  of  the 
funnel  with  a  watch  glass.  Boil  the  mixture  gently  for  two  hours. 
A  deep  straw  yellow  solution  should  be  obtained.  It  should  not  turn 
appreciably  blue  when  a  sample,  5  c.c.,  is  rendered  alkaline  with 
sodic  carbonate.  Dilute  to  a  liter. 

Preparation  of  Uric  Acid  Reagent.— Introduce  into  a  flask: 
750  c.c.  of  water, 
100  g.  of  sodium  tungstate, 

80  c.c.  of  phosphoric  acid  (85  per  cent.  H3PO4). 
Partly  close  the  mouth  of  the  flask  with  a  funnel  and  small  watch 
glass  and  boil  gently  for  two  hours.    Dilute  to  a  liter. 

Method  for  the  Determination  of  Chlorids  in  Blood  Plasma. — 

(Rappleye:  Jour.  Biol.  Chein.,  1918,  Vol.  32,  p.  509).  In  this 
method  the  principle  used  in  the  Volhard  Method  for  the  de- 
termination of  chlorids  in  urine  is  employed  for  the  estima- 
tion of  the  minute  amounts  of  sodium  chlorid  found  in  blood 
plasma. 

The  following  solutions  are  required: 

Solution  I  * 

Silver  Nitrate   7-2653  gm. 

Nitric  Acid   (concentrated)    250  c.c. 

Saturated  Solution  of  Iron-Ammonium-Alum 50  c.c. 

Distilled  water  to  make. 1000  c.c. 

Solution  II 

Potassium  sulphocyanate  in  distilled  water  of  such  strength 
that  25  c.c.  is  exactly  equivalent  to  5  c.c.  of  the  silver  nitrate  solu- 
tion. Each  c.c.  of  the  silver  nitrate  solution  is  exactly  equivalent 
to  2.5  mg.  of  sodium  chlorid  and  each  c.c.  of  the  potassium  sul- 
phocyanate is  equivalent  to  0.5  mg.  of  sodium  chlorid. 

Procedure. 

Place  2  c.c.  of  citrated  plasma  (oxalated  plasma  cannot  be 
used  on  account  of  the  poor  end  point)  in  a  50  c.c.  volumetric 
flask  containing  30  c.c.  distilled  water.  Add  10  c.c.  of  Solution 
I  and  make  to  mark.  After  being  mixed  the  liquid  is  allowed 
to  stand  for  5  to  10  minutes  and  is  then  filtered  through  a  dry 

207 


filter  paper  free  from  chlorids.  25  c.c.  of  the  filtrate  is  then 
titrated  with  Solution  II. 

To  calculate  the  result  subtract  the  number  of  c.c.  of  Solution 
II  used  in  the  titration  from  25  and  multiply  by  50.  This  gives 
the  number  of  milligrams  of  sodium  chlorid  present  in  100  c.c. 
of  blood  plasma. 

Nephelometric  Method  for  the  Determination  of  Fat  in  Blood 

(Bloor:  /.  Biol.  Chem.,  17,  377). — Run  3  c.c.  of  blood  slowly 
and  with  shaking  into  a  100  c.c.  volumetric  flask  containing  about 
80  c.c.  of  a  mixture  of  redistilled  alcohol  and  ether  (3:1).  Raise 
the  contents  of  the  flask  just  to  boiling  (with  constant  shaking) 
in  a  water-bath,  cool  in  running  water,  make  up  to  the  100  c.c. 
mark  with  more  alcohol-ether,  mix,  and  filter  into  a  small  flask 
or  bottle.  Stopper  tightly  as  soon  as  filtration  is  finished  to  avoid 
loss  of  liquid  by  evaporation. 

Measure  15  c.c.  of  the  filtrate,  containing  about  2  mg.  of  fat, 
into  a  small  beaker,  add  2  c.c.  of  N  sodium  ethylate,  and  evapo- 
rate the  mixture  just  to  dryness  on  the  water-bath.  To  the  dry 
residue  add  5  c.c.  of  alcohol-ether  (i  13),  and  warm  gently  until 
the  flakes  of  alkali  are  loosened  from  the  bottom  of  the  beaker. 
To  the  mixture  add  50  c.c.  of  water,  and  stir  until  a  clear  solu- 
tion is  obtained.  Add  5  c.c.  of  a  standard  solution  of  oleic  acid 
in  alcohol-ether,  containing  about  2  mg.  oleic  acid,  to  5°  c-c'  °f 
water  in  another  beaker.  To  the  standard,  and  to  the  blood  fat 
solution  add  (as  nearly  simultaneously  as  possible)  10  c.c.  of 
10  per  cent,  hydrochloric  acid.  Allow  the  suspensions  so  pro- 
duced to  stand  for  five  minutes,  and  then  compare  by  means  of 
the  Duboscq  colorimeter  previously  converted  into  a  nephe- 
lometer. 

For  the  comparison,  fill  the  two  nephelometer  tubes,  after  rins- 
ing with  the  solutions,  to  the  same  height  (the  meniscus  slightly 
above  the  dark  collar  at  the  top  of  the  tubes),  and  place  in  the 
nephelometer,  with  the  standard  tube  always  on  the  same  side. 
Set  the  movable  jacket  on  the  standard  tube  at  a  convenient  point 
(30  mm.  in  the  modified  colorimeter  described  below),  and  make 
comparisons  by  adjusting  the  jacket  on  the  test  solution  until  the 
images  show  equal  illumination.  Make  five  readings  alternately 
from  above  and  from  below,  and  take  the  average  as  the  reading. 
Make  the  calculations  in  the  same  way  as  in  the  colorimetric 
methods,  the  values  being  inversely  proportional  to  the  readings. 

209 


Changing  the  Duboscq  Colorimeter  into  a  Nephelometer. — (/. 

Biol.  Chem.,  22,  145). — A  simple  method  for  transforming  the 
Duboscq  colorimeter  into  a  nephelometer  is  described  in  the  Jour- 
nal of  Biological  Chemistry,  Vol.  22,  p.  145,  1915.  The  extra  parts 
necessary  are  supplied  in  an  improved  form  by  the  International 
Equipment  Company  of  Boston,  Mass.  By  the  use  of  these  parts 
the  change  may  be  quickly  made  as  follows:  Unscrew  the 
movable  glass  prisms  of  the  colorimeter,  slip  the  brass  collars 
for  the  nephelometer  tubes  into  place,  and  fasten  on  the  plate 
from  which  the  prisms  were  removed.  Slip  the  movable  jackets 
into  the  holes  in  the  cup  supports,  and  after  pushing  the  nephe- 
lometer tubes  into  place  in  the  collars,  the  instrument  is  ready 
for  use.  A  darkened  room  and  a  light-tight  box  for  the  light  are 
necessary.  The  box  should  be  about  48  cm.  long,  32  cm.  high, 
and  20  cm.  wide  for  the  ordinary  colorimeter.  It  should  contain 
a  bracket  at  one  end  to  support  the  light  (a  50  watt  "Mazda") 
at  the  height  of  the  nephelometer  tubes,  and  a  stop  at  the  other 
end,  against  which  the  instrument  may  be  pushed  and  so  placed 
that  the  nephelometer  tubes  are  about  30  cm.  from  the  light.  A 
slot  in  the  top  of  the  box  to  receive  the  telescope  of  the  instru- 
ment and  a  dark  curtain  to  cover  the  end  of  the  box  after  the 
instrument  is  pushed  into  place  complete  the  equipment  of  the 
box.  All  exposed  parts  should  be  painted  a  dull  black. 

Since  the  readings  obtained  from  suspensions  of  different 
strength  are  not  exactly  proportional  to  the  amount  of  precipitate 
present,  it  is  necessary  to  calibrate  the  instrument  for  different 
strengths  and  make  corrections  accordingly.  If,  however,  the 
solution  to  be  tested  is  within  25  per  cent,  of  the  value  of  the 
standard,  no  correction  is  necessary. 

A  Method  for  the  Determination  of  Cholesterin  in  Blood  or  Blood 
Serum. — The  method  consists  in  the  application  of  the  Auten- 
rieth-Funk  procedure  (Autenrieth  and  Funk — Munch,  med. 
Wochenschr.,  1913,  Vol.  69,  p.  1243)  to  the  alcohol-ether  extract 
of  blood  or  serum  prepared  as  for  the  determination  of  fat. 

Measure  10  c.c.  of  the  extract  into  a  small  beaker,  and  evapo- 
rate just  to  dryness  on  the  water-bath  or  electric  stove.  (Any 
heating  after  dryness  is  reached  produces  a  brownish  color,  which 
makes  the  determination  difficult  or  impossible.) 

Extract  the  cholesterin  from  the  dry  residue  by  boiling  out  3 
or  4  times  with  small  portions  (2-3  c.c.)  of  chloroform  and  de- 

211 


canting.  Evaporate  the  combined  extracts  to  a  little  less  than 
5  c.c.,  transfer  to  a  10  c.c.  graduated  cylinder,  and  make  the 
volume  up  to  5  c.c.  A  little  turbidity  does  not  matter,  since  it 
disappears  on  adding  the  reagents.  Measure  5  c.c.  of  a  standard 
cholesterin  solution  in  chloroform,  containing  .5  mg.  of  choles- 
terin,  into  a  similar  10  c.c.  graduate.  Add  to  each  2  c.c.  of  acetic 
anhydrid  and  .1  c.c.  of  concentrated  H2SO4.  Mix  the  solutions 
by  inverting  two  or  three  times,  and  set  the  cylinders  in  the  dark 
for  15  minutes;  then  transfer  the  solutions  to  the  colorimeter 
cups,  and  compare  as  usual,  setting  the  standard  at  15  mm. 

The  cement  of  the  colorimeter  cups  must,  of  course,  not  be  soluble 
in  chloroform.  Plaster-of-Paris  has  been  found  satisfactory,  or  even 
ordinary  glue,  if  the  cups  are  not  used  for  any  other  purpose. 


213 


INDEX 


Aceto-acetic  acid  in  urine,  159 
Acetone  in  urine,   151 
Acidimetry,    I 
Acidity  of  urine,  determination  of, 

127 

Acids,   strong  and  weak,   13 
Albumin    in    urine,     determination 

of,   171 

Albumins,   tests    for,   77 
Alkalimetry,  I 
Amino  acids,  83 
Ammonia,  determination  of,  23,  91, 

95 
Ammonia   in   blood,    determination 

of,  203 
Atom  weights,  3 

Benedict's   method    for   determina- 
tion   of    sugar,    53 
method     for     determination     of 

sugar    in    urine,    55 
reagent  for  sugar,  47 
standard   copper    solution,    55 
Beta-oxybutyric  acid  in  urine,   161 
Bile,   145 
Blood,  qualitative  experiments  with, 

135 
Bloor's     method     for     determining 

cholesterol  in  blood,  211 
nephelometric  method  for  fats  in 

blood,  209 
Bone,   143 

Carbohydrates,    47-69 

Calcium  in  urine,  determination  of, 

175 

Catalysis,  33-35 
Chlorids,  in  urine,,  determination  of, 

129-131 


Chlorids,  method  for  determining  in 

blood,  207 
Cholesterol,    Liebermann's    reaction 

for,  45 
method  for  determining  in  blood, 

211 

Colloids,    71-73 
Creatin,  in  urine,  117 
method  for  determining  in  blood, 

191 

Creatinin,  115 

determination  of,  in  urine,  117 
method  for  determining  in  blood, 

189 
Cystin,   preparation   of,   85 

Dextrin,  65 

Diacetic  acid  in  urine,   159 

Diastase,  65 

Fat,  39-45 

digestion,  43 

emulsification  of,  45 

method  for  determining  in  blood, 

209 
Fats,  iodin  number  of,  39 

saponification,   41 
Fatty  acids,  preparation  of,  41 

solubility  of,  39 

titration  of,  43 

Gelatin,  83 

Globulin,    test    for,   81 
Glycerin,   aerolein  test  for,  43 
Glycogen,  69 

Hippuric  acid,   119 

in    urine,    determination    of,    169 
Hydrochloric  acid,  test  for,  21 
Hydrogen-ion  concentration,   17 


215 


Indican  in  urine,   131 
Indicators,  5,  13 
Invert  sugar,  63 

Keratin,  83 

Lactic   acid,  test   for,  21 

Lecithin,  45 

Leucin,  83 

Levulose,  test  for,  51 

Magnesium,  in  urine,  determination 

of,   175 

Maltose,   preparation   of,   65 
Mass  Law,  37 
McCrudden's    method    for    calcium 

and  magnesium,   175 
Metabolism  experiments,  133 
Milk,  139 

sugar,  preparation  of,  65 
sugar   determination,   139 

Nephelometer,   211 
Nessler's  reagent,  203 
Nitrogen,    non-protein,    determina- 
tion of,  in  blood,  181 
determination,  colorimetric  meth- 
od for,  103 

in  ammonium  salts,  23 
Kjeldahl's   method,  25-31 
test  for,  in  protein,  75 

Pentoses,  test  for,  53 

Pepsin,  33 

Peptones,  83 

Phenol     determination     in     urine, 

167 

Phenol-uric  acid   reagent,    199,   205 
Phosphates  in  urine,   125 
Phosphoproteins,  83 
Phosphorus,  test  for,  in  protein,  75 
Potassium   in   urine,   determination 

of,  175 

Proteins,  71-85 
Proteoses,  83 


Reversible  reaction,  37 


2l6 


Shaffer's    method    for    beta-oxybu- 

tyric  acid   in   urine,    163 
Sodium  in  urine,  determination  of, 

175 
Solution,  equivalent,  I 

normal,  i 

standard  acetone,  155 

standard  ammonia,  103 

standard  creatinin,  117 

standard  hydrochloric  acid,  13 

standard    iodin,  153 

standard  oxalic  acid,  9 

standard  protein,  173 

standard  sodic  hydoxid,  n 

standard  sodium  thiosulphate,  153 

standard  uric  acid,  115 
Solutions,  colloidal,  71 
Sugar,    Benedict's    reagent    for,    47 

Benedict's  test  for,  47 

Folin's  test  for,  47 

fermentation  test  for,  53 

method  for  determining,  in  blood, 
197 

phenylhydrazin  test   for,  51 

Selivanoff's   test   for,  51 
Sugar  determination,   53-59 

polariscope   method,  61 
Starch  solution,  preparation  of,  65 
Sulphates,      determination     of,     in 

urine,   119-123 
Sulphur,  test  for,  in  protein,  75 

total,  determination  of,  in  urine, 
123 

Trypsin,  35 
Tryosin,    83 

Urea,  colorimetric  determination  of, 

in   urine,    107 
in  blood,   determination   of,    161- 

163 

reactions  with,   185 
Uric  acid  in  urine,  colorimetric  de- 
termination,   113 
murexid   test   for,    113 
phosphotungstic  acid  test  for,  113 
preparation    from    urine,    in 
Uric  acid  reagent,  207 
Urine,  89-133,  i5I-I77 

(3) 


NIVERS1TY  OF  CALIFORNIA  LIBRARY 

BERKET/BW 


Syracuse,  N.  Y. 

PAT.  JAN  21,  1908 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 


